Evaluation of basil extract ( Ocimum basilicum L.) on oxidative, anti-genotoxic and anti-inflammatory effects in human leukocytes cell cultures exposed to challenging agents

Abstract

Ocimum is one of the most important genera of the Lamiaceae family. Several studies about basil and its popular use reveal many characteristics of the herb, including its use as antioxidant, anti-aging, anti-inflammatory, anti-carcinogenic, anti-microbial, and cardiovascular agents, among others. In this paper, we evaluated genotoxic, oxidative, and anti-inflammatory parameters from the extract of Ocimum basilicum in different concentrations, using human leukocytes cultures exposed to challenging agents. Our results confirm that the O. basilicum extract acts as an antioxidant and effectively reverts or subjugates the effects of high oxidizing agents such as hydrogen peroxide. These actions are attributed to its composition, which is rich in polyphenols and flavonoids as well as compounds such as rosmarinic acid, all of which have well-known antioxidant activity. We also show that our basil extract presents anti-inflammatory properties, the mechanism of which is a composed interaction between the inhibition of pro-inflammatory mediator and the stimulation of anti-inflammatory cytokines. Although pharmacodynamics studies are necessary to evaluate the activities in vivo, our results demonstrated that basil could act as an antioxidant and anti-inflammatory and a possible alternative for medicinal treatment.

Full text

Braz. J. Pharm. Sci. 2017;53(1):e15098 Page 1 / 12
Brazilian Journal of
Pharmaceutical Sciences
http://dx.doi.org/10.1590/s2175-97902017000115098
Article
*Correspondence: M. M. Machado. Universidade Federal do Pampa. Campus Uru-
guaiana. BR 472, Km 585. Caixa Postal 118, 97500-970 – Uruguaiana - RS, Brasil.
E-mail: michelmachado@unipampa.edu.br
Evaluation of basil extract (Ocimum basilicum L.) on oxidative, anti-
genotoxic and anti-inflammatory effects in human leukocytes cell
cultures exposed to challenging agents
Camila Martins Güez1, Raul Oliveira de Souza2, Paula Fischer2, Maria Fernanda de Moura Leão2,
Jonathaline Apollo Duarte1, Aline Augusti Boligon2, Margareth Linde Athayde2, Luísa Zuravski1,
Luís Flávio Souza de Oliveira1, Michel Mansur Machado1,*
1Curso de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Pampa, Uruguaiana, Rio Grande do Sul,
Brasil, 2Departamento de Farmácia Industrial, Curso de Farmácia, Universidade Federal de Santa Maria, Santa Maria,
Rio Grande do Sul, Brasil
Ocimum is one of the most important genera of the Lamiaceae family. Several studies about basil and
its popular use reveal many characteristics of the herb, including its use as antioxidant, anti-aging, anti-
inammatory, anti-carcinogenic, anti-microbial, and cardiovascular agents, among others. In this paper, we
evaluated genotoxic, oxidative, and anti-inammatory parameters from the extract of Ocimum basilicum
in dierent concentrations, using human leukocytes cultures exposed to challenging agents. Our results
conrm that the O. basilicum extract acts as an antioxidant and eectively reverts or subjugates the eects
of high oxidizing agents such as hydrogen peroxide. These actions are attributed to its composition,
which is rich in polyphenols and avonoids as well as compounds such as rosmarinic acid, all of which
have well-known antioxidant activity. We also show that our basil extract presents anti-inammatory
properties, the mechanism of which is a composed interaction between the inhibition of pro-inammatory
mediator and the stimulation of anti-inammatory cytokines. Although pharmacodynamics studies are
necessary to evaluate the activities in vivo, our results demonstrated that basil could act as an antioxidant
and anti-inammatory and a possible alternative for medicinal treatment.
Uniterms: Ocimum basilicum/evaluation. Ocimum basilicum/genotoxic eects. Ocimum basilicum/
anti-inammatory properties. Medicinal plants/study.
INTRODUCTION
The free radicals are oxidizing agents, having one or
more unpaired electrons in their outer orbit, making them
highly reactive species that act as electrophiles (Wills,
Thomas, Gillham, 1989). The reactions of free radicals with
the organism result in cell and tissue damage that contributes
to the development of pathologies. The free radicals act on
cellular components by oxidizing lipids, proteins, nucleic
acids, and carbohydrates (Choi et al., 2002).
The genus Ocimum L. includes approximately
150 species, possessing a great variation in plant
morphology and biology, essential oil content, and
chemical composition (Danesi et al., 2008). Ocimum
basilicum, popular known as Basil or Sweet Basil, is a
common herb that belongs to Lamiaceae family. Studies
have shown many pharmacological effects in several
diseases, with potent antioxidant, anti-aging, anticancer,
antiviral, and antimicrobial properties (Sakr, Al-Amoudi,
2012). Studies have reported that rosmarinic acid (RA) is
the most biologically active compound present in Basil
related to this activities (Javanmardi et al., 2002; Lee,
Scagel, 2009; Shiga et al., 2009).
Traditionally, basil has been used as a medicinal
and aromatic herb, to add aroma and flavor to food
(Vieira, Simon, 2000) basil contains several secondary
metabolites like polyphenols, avonoids, and terpenes,
with recognized potential biologic eects that have been
identied in this species (Lee, Scagel, 2009). Researchers
have also tried to establish a standard of composition for

C. M. Güez, R. O. Souza, P. Fischer, M. F. M. Leão, J. A. Duarte, A. A. Boligon, M. L. Athayde, L. Zuravski, L. F. S. Oliveira, M. M. Machado
Braz. J. Pharm. Sci. 2017;53(1):e15098
Page 2 / 12
basil, both in the extract and in oil, but the existence of
more than 25 dierent types of O. basilicum, with many
constitutional dierences (Lee et al., 2005; Liber et al.,
2011) makes the task impossible.
The economic importance and global dissemination
of basil, with its many uses in cooking and folk medicine,
make it important to investigate its pharmacological and
toxicological eects in order to ensure its ecacy and
safety. Toxicity studies for these species are rare and do
not focus on genetic aspects of toxicity to human cells.
The main purpose of the present study was to evaluate the
oxidative, genotoxic, and anti-inammatory parameters
that may be present in the extract from Basil leaves using
human leukocyte cell cultures.
MATERIAL AND METHODS
Chemical, apparatus and general procedures
All chemicals were of analytical grade. Methanol,
acetic acid, gallic acid, chlorogenic acid, and caeic acid
purchased from Merck (Darmstadt, Germany). Quercetin,
rutin, rosmarinic acid, and kaempferol were acquired
from Sigma Chemical Co. (St. Louis, MO, USA). High
performance liquid chromatography (HPLC-DAD) was
performed with a Shimadzu Prominence Auto Sampler
(SIL-20A) HPLC system (Shimadzu, Kyoto, Japan)
equipped with Shimadzu LC-20AT reciprocating pumps
connected to a DGU 20A5 degasser with a CBM 20A
integrator, SPD-M20A diode array detector, and LC
solution 1.22 SP1 software.
Plant material
The commercial dry leaves of O. basilicum L., variety
Genovese (Chá & Cia Produtos Naturais®, Jacareí, SP),
were purchased from a local market in Uruguaiana, RS,
Brazil (latitude 29°45’17’’; longitude 57°05’18’’). The
leaves were triturated and macerated at room temperature
in hydroalcoholic solution (30 H2O: 70 ethanol v/v) at
concentration of 20 g per 100 mL of solvent for one week
under daily shaking. The maceration process was repeated
for two more weeks to exhaustion of the vegetable material.
At the end of three weeks, the filtrates were pooled and
evaporated under reduced pressure in a rotary evaporator
in order to remove ethanol and water. The dry extract of O.
basilicum (ObE) was used in the following tests.
Determination of total polyphenolic contents
The total polyphenol concentration in the crude
extract was measured spectrophotometrically using a
modied Folin-Ciocalteau method (Chandra, Gonzalez
De Mejia, 2004). Briey, we added 0.5 mL of 2 N Folin-
Ciocalteau reagent to 1 mL of each sample (0.15 mg/mL)
and allowed this mixture to stand for 5 min before adding
2 mL of 20% Na2CO3. Next we allowed the solution to
stand for 10 min before reading it in a Shimadzu-UV-1201
spectrophotometer (Shimadzu, Kyoto, Japan) at 730 nm.
We estimated the phenolic compounds in the crude extract
in triplicate. Because of the chemical heterogeneity of
plant products and the specicity of phenolic reagents, it
is very dicult to choose suitable standards for identifying
all phenols in a plant extract. Aqueous solutions of gallic
acid in the range of 0.001-0.2 mg/mL as references was
used. Thus, it is only possible to get relative equivalents
with the standard used. The total polyphenol content was
expressed as milligram equivalents of gallic acid per
milliliter of the extract.
Determination of total flavonoids
Methanolic solutions of quercetin in the range of
4.0–12.0 µg/mL as references was used. To 2 mL of each
reference solution, we added 20 mL of methanol and 1 mL
of 5% methanolic solution of AlCl3 (w/v) and produced
a total volume of 50 mL with methanol at 20 ºC. After 30
min, we measured the absorbance in a Shimadzu-UV-1201
spectrophotometer at 425 nm. We used the same procedure
to analyze the crude extract. The blank was 5% AlCl3 (w/v)
(Woisky, Salatino, 1998). We estimated the avonoids in
crude extract in triplicate. The contents were expressed
as milligram equivalents of quercetin/ mL of the extracts.
High performance liquid chromatography
Apparatus: The extracts were examined with high
performance liquid chromatography (HPLC) using an
HPLC system (Shimadzu, Kyoto, Japan), Prominence auto
sampler (SIL-20A), which was equipped with Shimadzu
LC-20 AT reciprocating pumps connected to a degasser
DGU 20A5 with an integrator CBM 20A, a UV-VIS
detector DAD (diode) SPD-M20A, and the Software LC
solution 1.22 SP1.
Analysis of phenolic compounds
Reverse phase chromatographic analyses were
performed under gradient conditions using a C18 column
(4.6 mm × 150 mm) packed with 5-μm diameter particles.
The mobile phase was water containing 2% acetic acid
(A) and methanol (B). The composition gradient was 5%
of B to 2 min, when it was changed to obtain 25%, 40%,

Evaluation of basil extract (Ocimum basilicum L.) on oxidative, anti-genotoxic and anti-inammatory eects in human leukocytes cell cultures
Braz. J. Pharm. Sci. 2017;53(1):e15098 Page 3 / 12
50%, 60%, 70%, and 100% B at 10, 20, 30, 40, 50, and
80 min, respectively, following the method described
by Laghari et al. (2011) with slight modications. The
extracts were analyzed at a concentration of 0.150 mg/
mL. We investigated the presence of the following six
antioxidants: gallic acid, chlorogenic acid, caeic acid,
quercetin, rutin, and kaempferol. These compounds were
identified by comparing their retention times and UV
absorption spectra with those of the commercial standards.
The ow rate was 0.7 mL/min and the injection volume
was 40 μL. The wavelengths were 254 nm for gallic acid;
327 nm for caffeic and chlorogenic acids; and 365 nm
for quercetin, rutin, and kaempferol. The samples and
mobile phase were ltered through a 0.45-μm membrane
lter (Millipore) and then degassed in an ultrasonic bath
before use. Stock solutions of reference standards were
prepared in the HPLC mobile phase for quercetin, rutin,
and kaempferol at 0.020–0.200 mg/mL and for gallic,
caffeic, and chlorogenic acids at 0.050–0.250 mg/mL.
A chromatography peak was conrmed by DAD spectra
(200–500 nm) and by comparing its retention time with
that of a reference standard.
Cytotoxicity curve in leukocytes
Initially, the dose-effect cytotoxicity curve was
determined in leukocytes using ObE dissolved in PBS
Buffer pH 7.2 at doses ranging from 0.0001 mg/mL to
100 mg/mL, to determine the lethal dose 50% (LD50).
Human leukocytes cultures were prepared using 0.5 mL
of venous blood collected by venipuncture from a male
volunteer (survey approved by the Ethics Committee of
the Federal University of Santa Maria, approval letter
number 23,081). They were centrifuged to separate
leukocytes and immediately transferred to RPMI 1640
medium supplemented with 10% fetal bovine serum,
1% streptomycin/penicillin, and phytohemagglutinin
according to a previous study described by Montagner et al.
(2010). Cells were kept at 37 °C for 72 h. The analyzed
parameter was cell proliferation according to Burow et al.
(1998).
Leukocytes culture sample preparations
To assess the anti-oxidative status and anti-
genotoxic proles, the leukocyte cultures were divided
in six groups. The groups were a negative control
(phosphate buer pH 7.2); a positive control (hydrogen
peroxide 100 μM); a group with rosmarinic acid at
the concentration previously found in the plant by the
phytochemical analysis; and three groups with dierent
extract concentrations obtained from toxicity studies;
that is, the LD50, LD50/10, and LD50/100. Groups with
rosmarinic acid and extracts have also received 100 µM
H2O2 to induce oxidation. To assess the inammatory
parameters the same groups division described above
were used, with exception of positive control that has
incorporated ibuprofen 100 μM. Additionally, all groups
except the negative control received dextran 1% to
induce the inflammatory process. The concentrations
were selected to establish effective doses and exhibit
low toxicity to leukocytes. All tests were performed in
triplicate.
Evaluation of antioxidants parameters of ObE in
human leukocytes cultures
To analyze the oxidative parameters, we used
classical techniques as lipid peroxidation, protein
carbonylation, ascorbic acid content, and superoxide
dismutase and catalase activities. The assays were
conducted in triplicates.
Lipid peroxidation
The extent of lipid peroxidation was estimated
as the concentration of thiobarbituric acid reactive
products (malondialdehyde) according to Ohkawa,
Ohishi, Yagi (1979). The method spectrophotometrically
measures the color produced by the reaction of TBA with
malondialdehyde (MDA) at 532 nm.
Protein carbonylation
The protocol was performed according to Morabito
et al. (2004). In this technique, carbonyl (CO) groups
(aldehydes and ketones) are produced on protein
side chains when they are oxidized, reacting with
2,4-dinitrophenylhydrazine, forming a color complex.
Ascorbic acid content
According to the method of Jacques-Silva et al.
(2001) the curve of ascorbic acid was taken as a reference
and the reference samples were mixed with trichloroacetic
acid 13.3% and 2,4-dinitrophenylhydrazine. After
incubation period, the samples were measured at 520 nm
in spectrophotometer. The extracts were measured using
the same procedure.
Catalase activity
Catalase activity was determined from the rate of
decomposition of H2O2 (Aebi, 1984). One unit of catalase
activity was dened as the required activity to degrade one
mol of hydrogen peroxide in 60s.

C. M. Güez, R. O. Souza, P. Fischer, M. F. M. Leão, J. A. Duarte, A. A. Boligon, M. L. Athayde, L. Zuravski, L. F. S. Oliveira, M. M. Machado
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Superoxide dismutase activity
Superoxide dismutase (SOD) (E.C.1.15.1.1) activity
was measured spectrophotometrically according to Boveris,
Cadenas (1997). The technique is based on the inhibition
of the reaction of superoxide anion with epinephrine. The
oxidation reaction of epinephrine produces adrenocromo,
which can be detected spectrophotometrically (480 nm).
The enzyme activity was determined by measuring the
rate of formation of adrenocromo. The reaction medium
contains glycine-NaOH and epinephrine. A unit of enzyme
activity is dened as the amount of enzymes required to
inhibit the rate of epinephrine autoxidation by 50%.
Evaluation of anti-genotoxic parameters of ObE in
human leukocytes cultures
The techniques used to evaluate the anti-genotoxic
parameters were cell proliferation and unviability, DNA
damage, chromosomal instability, and micronuclei
frequency.
Cellular proliferation and viability
Viability is assessed by the loss of membrane
integrity, using trypan blue (Burow et al., 1998). In this
technique, the same samples and respective concentrations
are combined with Turk’s solution (acetic acid 3% plus
gentian violet 1% in water), and after three min, the sample
is placed in a Neubauer chamber. The dierentiation of
living and dead cells is observed by the blue coloration of
dead cells. A total of 300 cells are counted and the amount
of total leukocytes (proliferation) is achieved through
counting in a Neubauer chamber.
Alkaline comet DNA assay
This test was assayed following procedures of Singh
et al. (1988). After incubation, the samples (leukocytes)
were mixed with low-melting point agarose and placed on
a microscope slide pre-coated with normal melting point
agarose. The slides were immersed in a lysis solution, and
an electrophoresis was performed (20 min at 300 mA and
25 V). In the end, the slides were neutralized and left to
dry overnight at room temperature. The dry slides were
re-hydrated and then xed for 10 min, and allowed to dry
again. The last stage was the coloring and the use of stop
solution. The slides were analyzed under blind conditions.
DNA damage was given as DNA damage index (DI).
The DNA damage was calculated from cells in dierent
damage classes.
Chromosomal instability
Colcemid was added in each leukocyte culture and
incubated at 37 °C during 60 min. After this, the cells were
centrifuged at 1,800 rpm for 10 min. The cell pellets were
re-suspended in hypotonic solution and incubated at 37 °C
for 16 min. After a new centrifugation, the cell pellets were
re-suspended in acetic acid: methanol (3:1) and poured
into a ask containing a xative solution (acetic acid and
methanol 3:1) followed by centrifugation. The slides were
prepared by dropping 3 or 4 drops of cell suspension into
a cold, wet slide, which then was air dried. The cells were
analyzed with 10× magnication to verify the density and
distribution of metaphase chromosomes (Yunis, 1976).
Micronuclei frequency
The cells were placed in a conic tube with saline
and centrifuged in 1,000 rpm for 5 min (this procedure
was repeated). One milliliter with the cell pellet was kept
in the tubes mixed with the pipette and spread over the
slide (two per sample) and left to dry in room temperature.
Slides were stained by panoptic method and then analyzed
under optical microscopy in immersion lens. For each
slide, 1,000 cells were counted (Schmid, 1975).
Evaluation of anti-inflammatory parameters of
ObE in human leukocytes cultures
For the determination of tumor necrosis factor–α
(TNF-α), interleukine-10 (IL-10), interleukine-6 (IL-6),
inhibition of COX-2 activities, and nitric oxide production,
measurements were made using ELISA kits according to
the manufacturer’s instructions. All tests were performed
in triplicate. The results of these tests were expressed
in percentage of production in relation to the negative
control.
Statistical analysis
Data were expressed as mean ± standard deviation
(SD). Comparisons between groups were performed using
one-way analysis of variance (ANOVA), followed by post
hoc of Bonferroni for multiple comparison tests. Nonlinear
regression analysis was used to determine LD50. Results
were considered statistically signicant when p<0.05.
RESULTS AND DISCUSSION
Medicinal plants are rich sources of natural
antioxidants and represent a promising perspective in
the discovery of new drugs in the therapeutic arena.
Most members of the Lamiaceae family have exhibited
interesting biological effects due to their antioxidant
compounds (Schoeld, Mbugua, Pell, 2001).

Evaluation of basil extract (Ocimum basilicum L.) on oxidative, anti-genotoxic and anti-inammatory eects in human leukocytes cell cultures
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Phenolic compounds are broadly distributed in
the plant kingdom and are the most abundant secondary
metabolites found in plants (Komali et al., 1999). The key
role of phenolic compounds as antibacterial is emphasized
in several reports (Møller et al., 1999; Schoeld, Mbugua,
Pell, 2001). Flavonoids compounds occur naturally in
plant foods and are a common component of our diet.
Table I shows the data from analysis by HPLC-DAD
method (Rice-Evans, Miller, Paganga, 1996) to determine
the concentration of totals polyphenol and avonoids and
some compounds present in O. basilicum L, all described
in literature as biologically active drugs.
In Figure 1, we show the dose-effect curve. As
shown below, when the dose of the extract is increased,
the total number of leukocytes decreases, showing a
dose-dependent eect. It is important to note that in LD50
concentration (35.44 µg/mL), the cellular unviability and
DNA damage showed no signicant changes (data not
shown), which agrees with the results by Gomez-Flores
et al. (2008). This result allowed us to establish the doses
that were used in our protocols. Aiming to search for a dose
with high aectivities and low toxicity, we determine the
work doses for this protocol: 35.44 µg/mL (LD50), 3.544
µg/mL (LD50/10), and 0.3544 µg/mL (LD50/100).
The accumulations of free radicals in organs or
tissues are strongly associated with oxidative damages in
biomolecules and cell membranes. This can lead to many
chronic diseases, such as inammatory, cancer, diabetes,
aging, cardiac dysfunction, and other degenerative
diseases (Wang et al., 2004). Figure 2 demonstrates the
results found for oxidative parameters.
The involvement of reactive oxygen species in
interactions with polyunsaturated fatty acids in cell
membranes generates lipid peroxidation. The result is the
formation of highly reactive hydro or lipoperoxides that
may trigger the oxidative cascade, resulting in damage
to membrane integrity (Tsang, Chung, 2009). The results
found in lipid peroxidation show that LD50 and LD50/10
obtained values are close to the control of rosmarinic
acid, a well-known antioxidant agent. Moreover, the
values of peroxidation in LD50 3,576 nMol of MDA/
mL in erythrocytes and LD50/10 4,226 nMol of MDA/
mL in erythrocytes, were 41.81% and 31.29% lower
when compared to the positive control, respectively. The
content of protein carbonyl is the most general indicator
and the most commonly used marker of protein oxidation,
TABLE I - Concentrations of some biologically important groups
and compounds presents in the samples used of O. basilicum L
Group / Compound Concentration in µg/mL of
Extract
Polyphenol compounds 23780.00 ± 145.30
Total avonoids 15982.00 ± 341.61
Quercetin 558.37 ± 2.41
Rutin 398.49 ± 0.97
Gallic acid 2330.52 ± 81.19
Caeic acid 4780.00 ± 14.52
Chlorogenic acid 2875.00 ± 103.56
Rosmarinic acid 353.00 ± 2.87
Kaempferol 342.00 ± 18.79
Data are expressed as means ± S.D. Results were conrmed by
an experiment that was repeated three times.
FIGURE 1 - Dose-eect curve using the crude extract of Ocimum basilicum in dierent doses (0.0001 mg/mL to 100 mg/mL)
to determine the lethal dose 50 (LD50) in human leukocytes. The inset shows the total amount of leukocytes versus the tests
concentrations of O. basilicum extract.

C. M. Güez, R. O. Souza, P. Fischer, M. F. M. Leão, J. A. Duarte, A. A. Boligon, M. L. Athayde, L. Zuravski, L. F. S. Oliveira, M. M. Machado
Braz. J. Pharm. Sci. 2017;53(1):e15098
Page 6 / 12
and its accumulation has been observed in several human
diseases. The values found in protein carbonyl show that
LD50 was 28% and LD50/10 18.8% lower when compared
to the positive control, and approximate to negative control
and rosmarinic acid control. Vitamin C or ascorbic acid
has been implicated in dierent biological processes and
plays an important role in oxidant defense. Ascorbic acid
functions in several enzymatic steps, acting like a cofactor
in the synthesis of collagen, monoamines, amino acids,
peptide hormones, and carnitine. Samples with higher
concentrations of extract showed higher concentrations
of ascorbic acid. This fact is directly related to the
concentrations of polyphenols and antioxidant action.
The most concentrated samples, LD50 and DL50/10, have
FIGURE 2 - Eects of O. basilicum extract in oxidative parameters of human leukocytes in culture subject to hydrogen peroxide.
In A: lipid peroxidation; B; carbonyl contents; C: ascorbic acid; D: catalase activity; E: superoxide dismutase activity. NC:
negative control; PC: positive control; RA: rosmarinic acid; LD50: concentration equals to LD50 (35.44 µg/mL); LD50/10: 10 times
less the concentration of LD50 (3.544 µg/mL); LD50/100: One hundred times less the concentration of LD50 (0.3544 µg/mL). Data
are expressed as mean ± S.D. Results were conrmed by an experiment that was repeated three times. Dierent letters represent
statistically dierent results among columns (p<0.05).

Evaluation of basil extract (Ocimum basilicum L.) on oxidative, anti-genotoxic and anti-inammatory eects in human leukocytes cell cultures
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higher levels of polyphenols. Polyphenols are a rst line
of defense against oxidative action of hydrogen peroxide.
When polyphenols decrease their concentrations, other
non-enzymatic antioxidants come into play, thereby
reducing the concentration of vitamin C (Kelm et al., 2000;
Petersen, Simmonds, 2003).
Mammalian cells have elaborate antioxidant defense
mechanisms to control damage eects of reactive oxygen
species and the catalase enzyme is one of these, protecting
cells against the toxic eects of hydrogen peroxide (Goyal,
Basak, 2010). The results of catalase analysis show the
ability of O. basilicum extract in neutralizing the eects
of hydrogen peroxide. The results were dose-related and
show a better eect at LD50 concentration. Superoxide has
been implicated in reactions associated with aging and in
pathophysiological processes due to its transformation
into more reactive species such as hydroxyl radical, which
initiates lipid peroxidation.
According to our results, the activity of SOD
enzyme was not aected in the three concentrations of
O. basilicum extract. All these results are related to the
presence of antioxidant compounds present in the extract
(Table I), as the groups of polyphenols and avonoids.
This information confirms the data from the literature
that phenolic compounds, especially flavonoids act in
the oxidative metabolism, not by changing the enzymatic
defenses, but by directing neutralizing of reactive species
in order to stabilize them (Liu et al., 2008).
We also evaluated the eects of hydrogen peroxide
and the counter eects of O. basilicum extract on human
leukocytes cells. The results of these markers are show
in Figure 3.
In cellular proliferation (Figure 3A), cellular increase
of the concentrations of LD50 and LD50/100 was similar to
the rosmarinic acid control and dierent from the positive
control, whereas the lowest concentration LD50/100 presented
a decrease in cellular proliferation, which may be compared
to the positive control. Figure 3B shows the percentage
of unviable cells for both controls groups and for the
three concentrations groups tested. The negative control
presented only 0.667%, whereas positive control was 14%
of unviable cells. Rosmarinic acid was 1.33% and the other
three concentrations of O. basilicum: LD50: 1.667%; LD50/10:
3.67%; LD50/100: 10.67% of unviable cells. Shimizu et al.
(2013) in a pancreatic cancer model, shows that Ocimum
sanctum, belonging to the same plant family tested here,
leaves inhibit the proliferation, migration, invasion, and
induce apoptosis of cells in vitro. It is important to note
that these results were obtained in cancer cells and it would
be inaccurate to extrapolate this statement for normal
leukocytes, having a functional metabolism.
According to Collins et al. (2008), the high
viability of cells is required as a previous condition
for the performance of the comet assay. Chromosomal
instability results in numerical and structural chromosomal
complexity and several studies associated these instabilities
with poor prognosis in solid tumors (Birkbak et al., 2011).
The results demonstrate that all concentrations
of extract of basil have presented percentages of
chromosomal abnormalities (Figure 3C) similar to the
negative control, and even the lowest concentration
presented 33.334% less mitotic index when compared to
positive control.
The comet assay is one of the most promising
genotoxicity tests developed to measure and analyze
DNA damage in single cells (Mukhopadhyay et al., 2004).
The test was used as a parameter for assessing the DNA
damage index (Figure 3D). As we can see at the lowest
concentration, LD50/100, the cells presented the highest
damage index compared with positive control, whereas the
other two concentrations of extract, LD50 and LD50/10,
showed damage indexes of 44.45% and 19.18% lower
when compared with the positive control, respectively.
The micronuclei assay provides a convenient
and reliable index of both chromosome breakage and
chromosome loss. The micronuclei are expressed in
dividing cells that either contain chromosome breaks
lacking centromeres (acentric fragments) and/or whole
chromosomes that are unable to migrate to the poles during
mitosis (Fenech, 2000). The results show the micronucleus
frequency (MN) found at the control groups and three
concentrations of plant extract under analysis (Figure
3E). The frequency of MN was dose-dependent. The
lowest concentration of the extract has shown the highest
frequency of cells with this alteration.
Basil was frequently reported as a common anti-
inammatory (Narayanaswamy, Duraisamy, Balakrishnan,
2011). Aiming to conrm this activity and evaluate the
mechanism involved, we perform a series of tests in human
leukocytes cell exposed to a pro-inammatory agent. The
results are presented in Figure 4.
Cytokines are subdivided in proinflammatory
(initiate defense against pathogens) and anti-inammatory
(regulate the inflammatory process helping to balance
the inflammatory response) acting with an important
role in inflammatory response. The proinflammatory
cytokines includes IL-1, IL-2, IL-6, IL-8, and TNF-α,
and the anti-inflammatory cytokines includes IL-1
antagonist receptor, IL-4, IL-10, and IL-13 (Goldstein,
Leung, Silverstein, 2006). The results show that the
production of the proinflammatory cytokines such as
TNF-α and IL-6 was not affected in the three different

C. M. Güez, R. O. Souza, P. Fischer, M. F. M. Leão, J. A. Duarte, A. A. Boligon, M. L. Athayde, L. Zuravski, L. F. S. Oliveira, M. M. Machado
Braz. J. Pharm. Sci. 2017;53(1):e15098
Page 8 / 12
doses of O. basilicum extract. However, the percentage
of production of IL-10, the anti-inammatory cytokine,
shows an increase in the percentage of more than 60% at
the highest dose of the extract when compared with the
positive control (ibuprofen). The percentage of production
of IL-10 also demonstrated to be dose-dependent, but in
all concentrations, this production was higher than the
negative and positive controls. The cyclooxygenases
(COX) have been identied in three dierent isoforms.
COX-2 acts like the inducible isoform, which is regulated
by growth factors and dierent cytokines (IL1β, IL6, or
TNFα) (Sobolewski et al., 2010). The ability of the extract
from O. basilicum to inhibit COX-2 was determined and
the results show that the extract was not capable to reduce
the activity of the cyclooxygenase like the positive control
(ibuprofen) but his activity decrease when compared to
FIGURE 3 - Eects of O. basilicum extract in anti-genotoxic parameters of human leukocytes in culture subject to hydrogen peroxide.
In A: cell proliferation; B; cell inviability; C: percentage of abnormal chromossomics; D: DNA damage index; E: frequency of
micronuclei. NC: negative control; PC: positive control; RA: rosmarinic acid; LD50: concentration equals to LD50 (35.44 µg/
mL); LD50/10: 10 times less the concentration of LD50 (3.544 µg/mL); LD50/100: One hundred times less the concentration of
LD50 (0.3544 µg/mL). Data are expressed as mean ± S.D. Results were conrmed by an experiment that was repeated three times.
Dierent letters represent statistically dierent results among columns (p < 0.05).

Evaluation of basil extract (Ocimum basilicum L.) on oxidative, anti-genotoxic and anti-inammatory eects in human leukocytes cell cultures
Braz. J. Pharm. Sci. 2017;53(1):e15098 Page 9 / 12
the negative control (100%) and was dose-dependent. The
inhibition of COX-2 in LD50 and LD50/10 approximates
to the inhibition caused by rosmarinic acid and several
studies report many properties of rosmarinic acid
including cyclooxygenase inhibition (Kelm et al., 2000;
Petersen, Simmonds, 2003).
The percentage of nitric oxide generated was
measured and cells pretreated with the three plant
extracts showed a dose-dependent inhibition. The
negative control was considered 100%. The maximal
NO inhibitory effect was exhibited by O. basilicum at
dose of LD50. Comparing to the negative control, the
FIGURE 4 - Eects of O. basilicum extract in inammatory parameters of human leukocytes in culture subject to dextran solution. All
the results are presented as percentage in relation to the negative control. In A: tumoral necrose factor-α (TNF-α); B; interleukine-10
(IL-10); C: interleukine-6 (IL-6); D: Ciclooxigenase type 2 activity (COX2 activity); E: nitric oxide (NO) production NC: negative
control; PC: positive control; RA: rosmarinic acid; LD50: concentration equals to LD50 (35.44 µg/mL); LD50/10: 10 times less
the concentration of LD50 (3.544 µg/mL); LD50/100: One hundred times less the concentration of LD50 (0.3544 µg/mL). Data
are expressed as mean ± S.D. Results were conrmed by an experiment that was repeated three times. Dierent letters represent
statistically dierent results among columns (p < 0.05).

C. M. Güez, R. O. Souza, P. Fischer, M. F. M. Leão, J. A. Duarte, A. A. Boligon, M. L. Athayde, L. Zuravski, L. F. S. Oliveira, M. M. Machado
Braz. J. Pharm. Sci. 2017;53(1):e15098
Page 10 / 12
percentage of reduction was higher than 30% (34.67%).
In a similar study using three plants of Lamiaceae family,
O. basilicum extract demonstrated the higher content of
phenols and maximal levels of DNA protection and free
radical scavenging against toxicity induced by cadmium
chloride (Thirugnanasampandan, Jayakumar, 2011). The
results suggested that the anti-inflammatory activities
of these extracts could be explained, at least in part, by
their antioxidant properties. Rosmarinic acid was one
of the most abundant caffeic acid esters present in O.
basilicum (Jayasinghe et al., 2003). It has been related
that this compound has antioxidant, anti-HIV, and anti-
inflammatory or cyclooxygenase and lipoxygenases
inhibitory activities (Petersen, Simmonds, 2003).
CONCLUSIONS
It is possible to verify that O. basilicum extract acts
as an antioxidant and effectively reverts or subjugates
the effects of a high oxidizing agent such as hydrogen
peroxide. These actions are explained by its composition,
which is rich in polyphenols and flavonoids as well as
compounds such as rosmarinic acid, which have a well-
known antioxidant activity.
Our study shows that Basil extract possesses anti-
inflammatory properties, and the mechanism involved
is a composed interaction between the inhibition of
pro-inammatory mediator and the stimulation of anti-
inammatory cytokines.
Although pharmacodynamic studies are necessary
to evaluate the activities in vivo, our results demonstrated
that Basil could act as antioxidant and anti-inammatory,
becoming a possible alternative for medicinal treatment.
CONFLICT OF INTERESTS
The authors declare no conict of interests.
ACKNOWLEDGMENTS
The authors are thankful to the financial support
of Fundação de Amparo a Pesquisa do Rio Grande
do Sul (FAPERGS) and to Conselho Nacional de
Desenvolvimento Cientíco e Tecnológico (CNPq) and to
Wiener Lab Group by the donation of kits for evaluations
of laboratory parameters.
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Received for publication on 28th May 2015
Accepted for publication on 18th November 2016