Rosmarinus officinalis L. (rosemary) as therapeutic and prophylactic agent

Abstract

Rosmarinus officinalis L. (rosemary) is a medicinal plant native to the Mediterranean region and cultivated around the world. Besides the therapeutic purpose, it is commonly used as a condiment and food preservative. R. officinalis L. is constituted by bioactive molecules, the phytocompounds, responsible for implement several pharmacological activities, such as anti-inflammatory, antioxidant, antimicrobial, antiproliferative, antitumor and protective, inhibitory and attenuating activities. Thus, in vivo and in vitro studies were presented in this Review, approaching the therapeutic and prophylactic effects of R. officinalis L. on some physiological disorders caused by biochemical, chemical or biological agents. In this way, methodology, mechanisms, results, and conclusions were described. The main objective of this study was showing that plant products could be equivalent to the available medicines.

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R E V I E W Open Access
Rosmarinus officinalis L. (rosemary) as
therapeutic and prophylactic agent
Jonatas Rafael de Oliveira
1*
, Samira Esteves Afonso Camargo
2
and Luciane Dias de Oliveira
1
Abstract
Rosmarinus officinalis L. (rosemary) is a medicinal plant native to the Mediterranean region and cultivated around
the world. Besides the therapeutic purpose, it is commonly used as a condiment and food preservative. R. officinalis
L. is constituted by bioactive molecules, the phytocompounds, responsible for implement several pharmacological
activities, such as anti-inflammatory, antioxidant, antimicrobial, antiproliferative, antitumor and protective, inhibitory
and attenuating activities. Thus, in vivo and in vitro studies were presented in this Review, approaching the therapeutic
and prophylactic effects of R. officinalis L. on some physiological disorders caused by biochemical, chemical or
biological agents. In this way, methodology, mechanisms, results, and conclusions were described. The main
objective of this study was showing that plant products could be equivalent to the available medicines.
Keywords: Rosmarinus officinalis L., Rosemary, Biological activities, Phytotherapy, Therapeutic effects,
Prophylactic effects
Background
Phytocompounds and pharmacological activities
R. officinalis L., popularly known as rosemary, is a plant
belonging to the family Lamiaceae and originated from
the Mediterranean region. However, it could be found
all over the world. It is a perennial and aromatic plant,
shrub-shaped with branches full of leaves, having a
height of up to two meters and green leaves that exude
a characteristic fragrance. R. officinalis may be used as
a spice in cooking, as a natural preservative in the food
industry, and as ornamental and medicinal plant [1–4].
Several phytocompounds presenting pharmacological
activities may be isolated from essential oils and extracts
of R. officinalis L. (Fig. 1), varying the concentration of
these molecules in each specimen of the plant. The phyto-
compounds most reported include caffeic acid, carnosic
acid, chlorogenic acid, monomeric acid, oleanolic acid,
rosmarinic acid, ursolic acid, alpha-pinene, camphor, car-
nosol, eucalyptol, rosmadial, rosmanol, rosmaquinones
A and B, secohinokio, and derivatives of eugenol and
luteolin [5–8]. Pharmacological effects of phytocom-
pounds from R. officinalis L. were showed in Table 1.
R. officinalis L. can promote several pharmacological
effects due to the interaction between the molecules of
the plant and the organic systems. The effects demon-
strated by this plant include (1) ability to attenuate asthma,
atherosclerosis, cataract, renal colic, hepatotoxicity, peptic
ulcer, inflammatory diseases, ischemic heart disease [9,
10]; (2) antioxidant and anti-inflammatory actions of ros-
marinic acid [11,12]; (3) control of hypercholesterolemia
and oxidative stress and relief of physical and mental fa-
tigue [13]; (4) myocardial blood pressure reduction with
rosmarinic acid [12]; (5) antiulcer action [14]; (6) lipid per-
oxidation reduction in heart and brain [15]; (7) antiangio-
genic and neuroprotective effects of carnosic acid and
carnosol [16]; (8) prevention of problems related to ath-
erosclerosis [17]; (9) anticancer and antiproliferative effects
[18–21]; (10) antiviral [22]; and antimicrobial actions [23];
(11) hepatoprotective [24], nephroprotective [25]and
radioprotective-antimutagenic capacities [26]; (12) gly-
cemia reduction [27]; (13) muscle relaxant and treatment
for cutaneous allergy [28]; (14) ability to treat depressive
behavior [29].
* Correspondence: jroliveira16@hotmail.com
1
Departamento de Biociências e Diagnóstico Bucal, Instituto de Ciência e
Tecnologia, Universidade Estadual Paulista (UNESP), Av. Engenheiro Francisco
José Longo, 777 –Jardim São Dimas, São José dos Campos, SP CEP
12245-000, Brazil
Full list of author information is available at the end of the article
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
de Oliveira et al. Journal of Biomedical Science (2019) 26:5
https://doi.org/10.1186/s12929-019-0499-8

Extraction methods
The extract of plant can be obtained from roots, stems,
leaves, flowers, fruits, seeds, and bark. Therefore, fresh
or dried samples can be used. However, according to
Vongsak et al. [30], a higher level of flavonoids was de-
tected in dried samples of Moringa oleifera leaves, as
compared to fresh samples.
Drying techniques include [31]:
a. Air-drying: a slower drying that can be performed
in a range of days, weeks and even months. The
process is conducted at room temperature exposing
the plant to the atmospheric air. In this way, those
unstable chemical compounds to the heat are not
damaged.
b. Microwave-drying: the drying time is faster than in
the air-drying process due to the electromagnetic
radiation. This process promotes collisions between
the molecules of the plant, resulting in heating that
causes water evaporation from the plant. Thus,
many phytocompounds can be denatured and lose
their pharmacological effectiveness.
c. Oven-drying: the drying time is also fast by using
heat to cause the water evaporation from the
plant. Unlike microwave-drying, in this process,
the phytochemicals are better preserved.
d. Freeze-drying: a drying performed using sublimation
method. The sample is initially frozen (−80 °C) for
12 h and immediately lyophilized. This method
favors the preservation of phytocompounds viability,
obtaining higher levels of these molecules than in
other drying methods.
Another relevant aspect is the size of the particles that
can interfere in the extraction process. Since, the smaller
the particle size, the higher the interaction between the
plant sample and the solvent to obtain the extract. Thus,
powder samples have better contact with the solvent
than crushed samples. Nanoparticles containing Centella
asiatica presented higher yields than microparticles,
when in contact with methanol [32].
During the extraction, the active part of the plant,
which contains the functional particles, is obtained, as
well as the residual part. The raw extracts are composed
of numerous active molecules, such as alkaloids, phen-
olic compounds, flavonoids, glycosides, and terpenoids.
From this initial extract, other types can be obtained by
various extraction methods, as can be observed in
Table 2[31].
The solvent to extract active compounds may interfere
with the final yield of these molecules. In Psidium gua-
java L. leaves extracts, the concentration of alkaloids,
carbohydrates, flavonoids, saponins, and tannins was
higher in ethanolic and hydroalcoholic solvents than pet-
roleum ether, chloroform or water [33]. The presence or
absence of some chemical constituents in the solvent
Fig. 1 Phytocompounds present in R. officinalis L
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 2 of 22

can interfere in the pharmacological activities; e.g., the
antioxidant activity was more prominent in Garcinia
atroviridis methanolic extract than in aqueous extract.
On the other hand, the aqueous extract of this plant
presented better antihyperlipidemic effect [34].
Table 1 Pharmacological effects of phytocompounds from R.
officinalis L reported in the literature
Phytocompound Pharmacological effect Reference
Caffeic acid a. Antibacterial [168]
b. Antioxidant [169]
c. Inhibitory effect of tumor cell
immigration
[170]
d. Inhibitory effect of tumor cell
proliferation
[171]
e. Protective effect of transplanted livers [172]
f. Apoptotic effect on tumor cells [173]
Carnosic acid a. Antiproliferative [174]
b. Protective effect of photoreceptor cells [175]
c. Antitumor [176]
d. Anti-inflammatory [177]
e. Inhibitory effect of digestive enzymes
(lipase, α-amylase, and α-glucosidase)
[178]
f. Suppressive effect of lipogenesis [179]
Chlorogenic
acid
a. Antioxidant [180]
b. Protective effect against lead-induced
renal damage
[181]
c. Protective effect against colitis [182]
d. Anti-infective [183]
Oleanolic acid a. Antiviral [184]
b. Protective effect against oxidative
stress-induced apoptosis
[185]
c. Antiproliferative [186]
d. Antitumor [187]
e. Antioxidant [188]
Rosmarinic acid a. Neuroprotective [189]
b. Protective effect against
nicotine-induced atherosclerosis
[190]
c. Complementary agent to the
anticancer chemotherapy
[191]
d. Anxiety control [192]
e. Antiproliferative [193]
f. Antiviral [194]
Ursolic acid a. Cytotoxic for tumor cells [195]
b. Anticancer [196]
c. Inducer of osteoblastic activity and
reducer of osteoclastic activity
[197]
d. Hypouricemic [198]
e. Proapoptotic [199]
f. Inductor of insulin sensitivity [200]
g. Protective effect against diabetic
nephropathy
[201]
h. Reducer of weight gain and
atherosclerosis
[202]
Alpha-pinene a. Antibacterial [203]
b. Antimicrobial [204]
Table 1 Pharmacological effects of phytocompounds from R.
officinalis L reported in the literature (Continued)
Phytocompound Pharmacological effect Reference
c. Protective effect against aspirin-induced
oxidative stress
[205]
d. Protective effect against peptic ulcer [206]
Camphor a. Immunomodulatory [207]
b. Antiproliferative [208]
c. Hypoglycemic [209]
d. Antimicrobial [210]
e. Antifungal, antihyphal, and antibiofilm [211]
Carnosol a. Antiproliferative [212]
b. Protective effect against renal
ischemia-reperfusion injury
[213]
c. Antifungal [214]
d. Proapoptotic and proautophagic [215]
e. Anti-inflammatory [216]
f. Anti-atopic dermatitis [217]
g. Antidiabetic [218]
Eucalyptol a. Proapoptotic [219]
b. Antibiofilm [220]
c. Control of infection and inflammation [221]
d. Anti-inflammatory [222]
e. Antinociceptive [223]
f. Antiviral [224]
Rosmanol a. Antinociceptive, antidepressant,
and anxiolytic
[82]
b. Anticancer [225]
Eugenol a. Acaricidal [226]
b. Antifungal [227]
c. Chemotherapeutic on cervical
cancer cells
[228]
d. Antiproliferative [229]
e. Anti-inflammatory and antioxidative [230]
Luteolin a. Anti-inflammatory [231]
b. Anti-atopic dermatitis [232]
c. Proapoptotic and proautophagic [233]
d. Antimicrobial [234]
e. Antiproliferative [235]
f. Protection of microglia against
rotenone-induced toxicity
[236]
g. Inhibitory effect of glucocorticoid-
induced osteoporosis
[237]
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 3 of 22

The chosen method for extraction of compounds may
interfere the yield of the sample. The use of high tem-
peratures (90 °C) for C. asiatica extraction provided an
increased yield of phenolic compounds which provided
better antioxidant activity [35]. However, in microwave
assisted extraction, a duplicate increase in the yield of C.
asiatica triterpene was detected, compared to the Soxh-
let extraction [36].
The purpose of the study
Because of the immense variety of beneficial effects that R.
officinalis L. has demonstrated, in vivo and in vitro studies
were addressed in this Review (Table 3). Therapeutic and
prophylactic effects of this plant on some physiological
disorders caused by biochemical, chemical or biological
agents were considered. This Review consisted of method-
ology, mechanisms, results, and conclusions of these stud-
ies. The main purpose of this work was demonstrating the
ability of a medicinal plant (R. officinalis L.) to treat health
problems, and showing its equivalence to any other medi-
cine, concerning its beneficial effects.
Cardiac remodeling after myocardial infarction
Background
Myocardial infarction is a condition characterized by the
cardiac muscle necrosis, due to cell death caused by in-
flammation, which may be initiated by oxidative stress
that produces cytokines synthesis, such as tumor necro-
sis factor-α(TNF-α) and interleukins (IL-1βand −6);
reabsorption of necrotic tissue; exacerbated collagen
deposition; and hypertrophy. Both reactive oxygen species
(ROS) and cytokines may induce the action of metallopro-
teinases (MMP), as well as the collagen accumulation,
which is responsible for causing changes in the size,
weight and function of the heart. Besides, the continued
presence of metabolites from affected cells may also pro-
vide these changes. Thus, a forced adaptation of the organ
to a new reality may occur, providing a cardiac remodeling
that could lead to heart failure [37,38]. In this manner,
the use of antioxidants has been evaluated in these cases.
Also, other types of medications have been used, such as
the vasodilators prazosin, diltiazem, and felodipine, show-
ing no satisfactory outcome regarding mortality reduction
or hospitalization [39–41]. Positive inotropic drugs, which
have a good hemodynamic effect, can also present signifi-
cant side effects, about patient survival due to the neu-
rohormonal activation and ventricular arrhythmias.
Milrinone, flosequinan, pimobendan, ibopamine, and ves-
narinone have caused an increase in mortality in chronic
heart failure [42–45]. To control the oxidizing agents, the
effectiveness of some natural products, including R. offici-
nalis L., has been investigated, mainly by the presence of
bioactive molecules with antioxidant capacities, such as
rosmarinic acid, carnosic acid, and carnosol [46].
Methodology
The effect of the supplementation with R. officinalis
L.leaves was evaluated on cardiac remodeling after
Table 2 Extraction methods [31]
Method Description
Maceration Powdered or crushed materials are left in solvents for at least three days at room temperature under
agitation. Them, the solution is filtered. Phytocompounds are released by breaking the cell wall of plant cells.
Infusion The same maceration process is used, but the period is shorter, and the sample is boiled in specific volumes
of water.
Decoction The same maceration and infusion processes are used, but the extractions of thermostable compounds and
substances from hard parts of the plant such as roots and bark are possible.
Percolation The same maceration and infusion processes are used. The sample is placed in contact with boiling water,
and the extraction is performed for about two hours. In the end, a concentrated extract is obtained.
Soxhlet extraction The extraction process is performed in the Soxhlet extractor. Sample and solvent are placed in the apparatus.
Upon heating the solvent, the solid particles from the substance are extracted. The generated liquid is absorbed
and filtered. A more concentrated sample is obtained, and the heating of the solvent does not harm
the compound.
Microwave assisted extraction Use of microwaves to reach the molecules in a sample inside the solvent. The heating generated on the surface
of the sample promotes changes in the structures of the chemical elements and favors the entry of the solvent
into the material and consequently the extraction of the compounds.
Ultrasound-assisted extraction Ultrasound (20 to 2000 kHz) is used for the extraction of the compounds. In this process, there is an increase in
solvent contact with the sample, due to increased permeability of the plant cell wall. Sound waves impair the
molecular integrity of the cell wall and thus favor the release of phytochemical agents.
Accelerated solvent extraction In an automated way, compounds are extracted from solid and semi-solid samples, using small volumes of solvents,
at high temperatures and pressures.
Supercritical fluid extraction This extraction is performed using supercritical fluids as solvents, both in solid and liquid samples. Carbon dioxide
(CO
2
) is the most commonly used fluid. This method can also be used for analytical purposes and the removal of
unwanted substances or separation of a particular phytocompound in a sample. Temperature and pressure should
be considered (~ 31 °C and 74 bar).
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 4 of 22

myocardial infarction in male Wistar rats [47]. For this,
healthy animals and infarcted animals were fed with
standard chow or chow containing 0.02% or 0.2% of R.
officinalis L. leaves for 90 days. The animals were evalu-
ated by transthoracic echocardiographic exam. Other
analyses were performed on the left ventricle from the
sacrificed animals, such as: (i) checking of the infarct
extension in size and length; (ii) muscular viability by
endocardial and epicardial circumferences; (iii) chem-
ical mediators levels, such as TNF-α, IFN-γ, IL-10,
MMP-2 and TIMP-1; (iv) total protein and lipid hydro-
peroxide levels; (v) enzymatic activity of glutathione
peroxidase, superoxide dismutase, catalase; and (vi) car-
diac metabolism checked by the activity of β-hydroxyacyl
coenzyme-A dehydrogenase, lactate dehydrogenase, cit-
rate synthase, Complex I (NADH:ubiquinone oxidoreduc-
tase), Complex II (succinate dehydrogenase), and ATP
synthase.
Findings
No deaths were observed in healthy animals; however,
among infarcted animals, two deaths were verified in
the groups that received standard chow and supple-
mentation with 0.02% of R. officinalis L. leaves, besides
one death in the group supplemented with 0.2%. Never-
theless, the infarction size was similar among infarcted
animals and was not found any difference between the
weight gain and systolic arterial pressure in all groups.
The infarction generated an adverse cardiac remodel-
ing, demonstrated by increased of left ventricular diam-
eter; high collagen percentage; alterations in the diastolic
and systolic functions; intensification of oxidative stress;
metabolic changes evidenced by modification of enzym-
atic activity; increased of MMP-2 activity and decreased of
IL-10, TNF-α,IFN-γlevels. However, the supplementation
with R. officinalis L. leaves improved diastolic function, re-
duced muscle hypertrophy, provided morphological and
functional changes in the heart of infarcted animals, veri-
fied by increased β-oxidation of fatty acids and reduced
lactate oxidation, besides improved respiratory chain per-
formance. R. officinalis L. significantly decreased oxidative
stress, even though the used concentrations provided dif-
ferent scenarios regarding the diastolic function and
hypertrophy, since the supplementation with 0.02% pre-
sented lower left atrium and supplementation with 0.2%
demonstrated higher Complex II activity. Additionally,
collagen percentage, cytokines levels, and MMP-2 activity
were not altered with any of the supplementations.
Table 3 Pharmacological effects of R. officinalis L. summarized in this Review
Pharmacological effect Product from R. officinalis L. Main findings Reference
Cardiac remodeling after
myocardial infarction
Supplementation with leaves a. Attenuation of cardiac remodeling
b. Improvement of metabolism and reduction of
oxidative stress
[47]
Control of body weight and
dyslipidemia
Aqueous extract a. Inhibition of the body weight gain
b. Scavenging of free radical
c. Inhibition of gentamicin (GM)-induced
hepatotoxicity
d. Antioxidant action
e. DNA-protective effect
[60]
Neuroprotective effect on
cerebral ischemia
Hydro-alcoholic extract a. Absence of dyslipidemia effect
b. Reduction of acute ischemic stroke lesion
[71]
Antinociceptive effect Ethanolic extract Pain control [77]
Mono- and polymicrobial
biofilms reduction
Glycolic extract a. Antimicrobial effect
b. Action on monomicrobial biofilms of C. albicans,
S. aureus,E. faecalis,S. mutans, and P. aeruginosa
b. Action on polymicrobial biofilms formed by
C. albicans with each bacterium
[91]
Hepato-nephrotoxicity inhibition
of the lead
Ethanolic extract a. Protection of structure and function of liver and
kidney against lead
b. Stabilization of antioxidant proteins
[123]
Stress relief in situation of
real danger
Hydro-alcoholic extract a. Anxiolytic effect
b. Stress control
[132]
Human tumor cells proliferation
inhibition
Glycolic extract Breast adenocarcinoma (MCF-7) and cervical
adenocarcinoma (HeLa)
[91]
Methanolic and ethyl
acetate extracts
Epithelial colorectal adenocarcinoma (CaCo-2)
and histiocytic lymphoma cell line (U-937)
[152]
Aqueous extract Esophageal squamous cell carcinoma (KYSE30)
and gastric adenocarcinoma (AGS)
[153]
Methanolic extract Lung carcinoma (A549) [154]
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 5 of 22

Action mechanisms
Molecular and cellular changes in the heart are respon-
sible for the clinical problems. Thus, metabolic pathways
and antioxidants could be the interaction forms of R.
officinalis L. with living tissue [47,48].
Metabolic changes, oxidative stress, and redox signal-
ing are factors that contribute to cardiac remodeling
[49]. Murino Rafacho et al. [47] demonstrated that ani-
mals submitted to myocardial infarction and supple-
mented with R. officinalis L. leaves showed a higher fatty
acids oxidation and respiratory chain improvement, simi-
lar to the metabolism of non-infarcted animals. Besides,
they found a decrease of oxidative stress and enzymatic
activity in cardiac tissue, using supplementation.
Oxidative stress, caused by the action of reactive oxy-
gen species (ROS), can be controlled by antioxidant en-
zymes such as superoxide dismutase and catalase [50].
The enzyme superoxide dismutase is the first to protect
the mitochondria against harmful effects of ROS during
cardiac remodeling [51]. According to Chohan et al. [52],
R. officinalis L. can function as an antioxidant enzyme and
remove superoxide radicals from the tissue.
Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is re-
sponsible for the transcription of genes encoding anti-
oxidant enzymes, and an increase in its expression has
been noticed after treatment with R. officinalis L. [53,
54]. Therefore, supplementation with this plant has
demonstrated an antioxidant characteristic comparable
to its healthy cells [47].
Control of body weight and dyslipidemia
Background
Lipid metabolism may be altered and lead to increased
levels of total cholesterol, low-density lipoprotein choles-
terol and triacylglycerols, in the blood, causing cardio-
and cerebrovascular disorders [55]. The use of some
medications may induce to dyslipidemia, including anti-
rheumatics [56], second-generation antipsychotics [57],
antiretrovirals [58], and antibiotics (gentamicin) [59]. In
this way, plant products to control dyslipidemia has been
investigated.
Methodology
The toxicity caused by gentamicin was attenuated in
Sprague-Dawley rats with administration of R. officinalis
L. extract [60]. In this study, the animals received genta-
micin by intraperitoneal injection and 8% R. officinalis L.
aqueous extract orally (10 mL/kg), the control groups
were treated with saline solution (0.9% NaCl) or genta-
micin (60 mg/kg). Doses were daily given over 10 days.
Findings
The body weight of the animals increased significantly in
the group treated with gentamicin compared to the
control group (saline), demonstrating that the antibiotic
could change the body mass. On the other hand, there
was an inhibition of the body weight gain using the
co-administration of the extract. Besides, the plant prod-
uct also significantly reduced the liver weight of the ani-
mals compared to the group treated with antibiotic. The
liver injuries caused by gentamicin were reversed with
the administration of the extract. The harmful effects of
this antibiotic were attenuated with plant extract at the
liver level, providing a significant decrease in alanine ami-
notransferase and aspartate aminotransferase activity and
total bilirubin levels. R. officinalis L. extract presented hy-
polipidemic effect, as evidenced by significant reductions
in total cholesterol, phospholipids, triacylglycerols and
atherogenic index. Additionally, the co-administration of
the extract also protected against DNA damage, demon-
strated by the absence of genetic material fragmentation
in treated animals.
Action mechanisms
Plants from Lamiacea family are rich in phytocom-
pounds, such as catechins, coumarins, and cinnamic
acid. These molecules are responsible for exerting sig-
nificant antioxidant activity, as well as quercitin, luteo-
lin, kaempferol, and rosmarinic, hydrocafeic and caffeic
acids [61]. Thus, R. officinalis L. can protect the organ-
ism against hyperlipidemic and hepatotoxic effects pro-
moted by some products, as gentamicin [60]. This
antibiotic can affect the liver and enhance the enzym-
atic activity of aspartate transaminase (AST) and ala-
nine transaminase (ALT), as well as increase the
bilirubin level and decrease the protein synthesis. Also,
gentamicin is responsible for increasing the levels of
triglyceride, cholesterol, and phospholipid, besides im-
proving the pancreatic lipase activity [60,62]. Hyperlip-
idemia can favor the emergence of heart disease and
contribute for an increase of body weight.
R. officinalis L. acts decreasing the hydrogen peroxide
levels, which promotes protection against oxidative stress
caused by a toxicity inducer, as gentamicin. In fact, the
plant can reduce the ROS production and protect the hep-
atic tissue from damage in DNA, proteins, and mem-
branes [60]. Additionally, R. officinalis L. can increase
the activity of phase I and II enzymes, providing a de-
toxification effect [63].
Regarding the dyslipidemia aspects, R. officinalis L. in-
hibits the activity of 3-hydroxy-3-methylglutaryl coenzyme
A(HMG-CO) reductase. This provides a significant chol-
esterol reduction by oxidative stress [60]. Yokozawa et al.
[64] reported that polyphenols can induce fecal excretion
of total cholesterol and bile acids. Thereby, a decreased
level of cholesterol in plasma can be observed because it is
used in the biliary juice synthesis. Besides, an absorption
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 6 of 22

reduction of this lipid in the intestine also is verified due
to the low disposition of bile acids.
Neuroprotective effect on cerebral ischemia
Background
The localized blood flow reduction in the brain is known
as cerebral ischemia, caused by arteries obstruction or
systematic hyperfusion, causing irreversible damage. In-
flammation and oxidative stress may be related to this
physiological disorder [65]. The unexpected decrease of
vital supplies (oxygen and nutrients), due to ischemia,
may lead to stroke [66], which is caused by the edema
from the rupture of the blood-brain barrier [67]. This li-
quid accumulation contributes to increase the brain
mass [68] and consequently promote cell death [69].
Thus, recombinant tissue plasminogen activator (TPA)
has been used for the treatment of this problem; however,
this product has caused a worsening of the lesions as a re-
sult of the cerebral ischemia, contributing to the increase
in the infarct size, cerebral edema, and hemorrhage intra-
cranial [70]. Thus, alternative products for the treatment
has been studied, including the plant products.
Methodology
Hydroethanolic extract obtained from the R. officinalis
L. leaves has been demonstrated in providing brain toler-
ance to artificially induced ischemia [71]. For this ana-
lysis, the authors promoted occlusion of the middle
cerebral artery with intraluminal nylon filament implant-
ation for 60 min in adult male Wistar rats, restoring
blood flow after this period. Moreover, the animals were
previously treated with the extract at 50, 75 or 100 mg/
kg/day or with the vehicle (control) for 30 days. Ischemia
induction occurred 2 hours before the last treatment.
Non-ischemic animals were also included in the study
for comparative purposes. The reperfusion period was
24 h and then the analyses were performed on the ani-
mals, including: (i) total cholesterol (TC), triglyceride
(TG), low density lipoprotein (LDL-c) and high density
lipoprotein (HDL-c) levels, quantified on the 30th day of
treatment before surgery; (ii) neurological functions
assessed by means of scores, such as 0 (“no neurological
dysfunction”), 1 (“failure to extend opposite forepaw”), 2
(“circling to the contralateral side, when held by tail with
feet on floor”), 3 (“falling to the left”), 4 (“unable to bear
weight on affected side”/“no spontaneous walking and a
depressed level of consciousness”), and 5 (“death”); and
(iii) neurological behavior, including volume of the in-
farct and edema and permeability of the blood-brain
barrier.
Findings
After the 30th day, all animals gained weight; however, it
was lower in the group treated with 100 mg/kg. R.
officinalis L. extract decreased TC, TG, and LDL-c and
increased HDL-c levels. In the treated groups were ob-
served low levels of LDL/HDL and TG/HDL after 30
days. These data demonstrated that R. officinalis L. ex-
tract had no dyslipidemic effect. Regarding the neuro-
logical functions, the plant extract contributed to
reducing the neurological deficit, since the untreated
group presented score 3 (“falling to the left”), and after
using doses of 50, 100 and 75 mg/kg the score was 1
(“failure to extend opposite forepaw”). The extract also
provided a reduction in the infarction volume, present-
ing an excellent protection in the groups treated with 75
and 100 mg/kg. On the other hand, in untreated groups,
the induced ischemia caused severe infarction in the
subcortex and cerebral cortex regions. The edema for-
mation was controlled in the animals treated with R. offi-
cinalis L. extract since protection against rupture of the
blood-brain barrier and non-extravasation of liquid were
observed.
Action mechanisms
Seyedemadi et al. [71] found that R. officinalis L. hydro-
ethanolic prevented the rupture of the blood-brain bar-
rier, as well as the cerebral edema, infarction, and
neurological problems, in a murine model with middle
cerebral artery occlusion. This can occur due to the abil-
ity of R. officinalis L. to prevent the mitogen-activated
protein kinase (MAPK) phosphorylation, which provides
the blockade of nuclear factor kappa B (NF-kB) activa-
tion. This blocking will decrease the expression of nitric
oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).
During the inflammatory process, leukocyte activity and
action of proinflammatory enzymes and other mediators,
such as nitric oxide (NO), interleukin 1 beta (IL-1β), and
tumor necrosis factor-alpha (TNF-α), can significantly
decrease [72]. In ischemia pathogenesis, the oxidative
stress is remarkable and can lead to the rupture of the
blood-brain barrier and neurons death [73]. According
to Huang et al. [74], R. officinalis L. can promote reduc-
tion of lipid peroxidation, hydroxyl radical, and hydro-
gen peroxide action in some tissues, such as cerebral,
renal, cardiac, and serum. This fact shows that the plant
can control the release of oxidative stress promoting
molecules which are harmful to brain health.
Antinociceptive effect
Background
Cyclooxygenase inhibitor medicines have been used to
treat pain, such as non-steroidal anti-inflammatory drugs
(NSAIDs), although the prolonged use of these medica-
tions may lead to cardiovascular, renal, and gastric com-
plications [75,76]. In contrast, products obtained from
medicinal plants can operate synergistically with these
medicines to control pain.
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 7 of 22

Methodology
The synergistic and antinociceptive activities of R. offici-
nalis L. ethanol extract was reported in a study con-
ducted in Wistar rats [77]. The animals were previously
treated with plant extract, phytocompounds (ursolic acid
and oleanolic acid), ketorolac, an NSAID, and ketorolac
associated with plant products. Nociception was induced
by subcutaneous injection of 1% formalin in the right
paw dorsum.
Findings
Plant extract (0.58 μg/paw) and ketorolac (0.88 μg/paw)
provided antinociception of 66.5%. Higher doses of these
products (10 μg) showed values of 38.5 and 42.6%, re-
spectively. Thus, the drug interaction presented more ef-
fective antinociceptive action using lower doses. The
administration of ursolic acid and oleanolic acid pro-
vided antinociception of 48.7 and 47.5%, respectively.
Additionally, the association of extract or ursolic acid
with ketorolac presented a nociception reduction of 61.1
and 71%, respectively. The phytocompound may be one
of the responsible for the synergistic and antinociceptive
effects of R. officinalis extract.
Action mechanisms
Antinociceptive activity can be increased with synergism
between NSAIDs and plant products, such as extracts
and phytocompounds from R. officinalis L. Thus, doses
of analgesics could be reduced, as demonstrated by Bel-
trán-Villalobos et al. [77] which treated rats with ketoro-
lac associated with R. officinalis L.
R. officinalis L. has caused inhibition of pain, according
to preclinical studies, due to its interaction with opioid
and 5-hydroxytryptamine (5-HT1A) receptors [78–80].
In inflammation model, R. officinalis L. essential oil
showed effective antinociceptive activity in association
with endogenous opioids in the serothogenic system, via
5-HT 1A receptor [79].
Lee et al., [81] found that eugenol, a phytocompound
from R. officinalis L., can act on γ-aminobutyric acid
type A (GABAA) receptor modulation in trigeminal gan-
glion neurons. Other compounds of this plant, such as
rosmanol, cirsimaritin, and salvigenin, have also shown
antinociceptive effect, by GABAA receptor modulation
[82]. Heperidine, obtained from R. officinalis L., has also
been induced inhibition of pain by interacting with transi-
ent receptor potential cation channel subfamily V member
1 (TrpV1) [83]. These authors also found that the inter-
action of hyperidin with ketorolac has shown a synergistic
antinociceptive effect on inflammatory pain. Other phyto-
compounds from R. officinalis L. such as α-phellandrene
and ursolic acid can also act on TrpV1 receptors [84,85].
The antinociceptive effect of ursolic acid is modulated by
cyclic guanosine monophosphate (cGMP) and 5-HT
1A
[85]. Poeckel et al. [86] found that phytocompounds from
R. officinalis L. can decrease ROS formation, inhibiting
5-lipoxygenase, COX-2, and leukocytes, and blocking
Ca
2+
channels in polymorphonuclear cells.
Mono- and polymicrobial biofilms reduction
Background
Biofilms are formed by microbial communities of differ-
ent species adhered to the biotic or abiotic substrate, be-
ing surrounded by polysaccharide extracellular matrix
produced by the microorganisms. This structure offers
protection to the microorganisms against the external
environment, actions of the host’s defense system and
antimicrobial agents [87,88]. The proportion of micro-
bial cells and extracellular matrix may range between 10
and 25% of cells and 75–90% of polymeric substances
[89]. The microorganism arrangement in these three-di-
mensional structures gives them about a thousand times
more antimicrobial resistance than in planktonic cells, be-
ing directly related to cases of infectious diseases [90].
Therefore, the development of new products or strategies
to combat microorganisms in biofilms is important. An-
other concern of the scientific community is the constant
emergence of antimicrobial-resistant strains, which has
been stimulating the search for alternative methods to
control pathogenic microorganisms.
Methodology
Phytotherapy is a wide field that can use plant products
as an antimicrobial. The results of some studies have
been increasingly promising and motivating. R. officinalis
L. glycolic extract is an example of this, since its ability
to control mono- and polymicrobial biofilms were cited
[91]. In this study, the authors proposed to evaluate the
effect of this plant extract on microorganisms that cause
oral infections, such as Candida albicans, responsible for
pseudomembranous/erythematous candidiasis and angu-
lar cheilitis [92]; Staphylococcus aureus, related to peri-
odontitis due to its presence in supra- and subgingival
biofilms [93]; Enterococcus faecalis, associated with
asymptomatic endodontic infections characterized by
formation of periapical lesions [94]; Streptococcus mutans,
one of the agents that promote the development of dental
caries [95]; and Pseudomonas aeruginosa linked to more
aggressive periodontitis [96]. According to de Oliveira et
al. [91], the action of R. officinalis L. extract was analyzed
both on monomicrobial biofilms of each species and poly-
microbial biofilms formed by C. albicans associated with
S. aureus,E. faecalis,S. mutans or P. aeruginosa. These
microbial associations were carried out once these species
cause important clinical manifestations or present peculiar
behavior when they are together. It has been reported that
C. albicans may favor the development of S. aureus [87]
and, besides, this bacterium was found in 27% of
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 8 of 22

candidemia in nosocomial infections [97]. The association
of C. albicans with E. faecalis both are benefited, and their
pathogenicity may decrease, unlike when they are alone
[98]. The interaction of C. albicans with S. mutans results
in an extremely virulent biofilm on the teeth [99]. The de-
velopment of C. albicans may be regulated by enzymes se-
creted by P. aeruginosa; these proteins affect the process
of cellular respiration and hyphal formation [100]. There-
fore, mono- and polymicrobial biofilms were formed in
microplate for 48 h. After, planktonic cells were discarded
by washes with saline solution (0.9% NaCl) and the bio-
films were treated with R. officinalis L glycolic extract
(200 mg/mL) for 5 min, considering its use as dentifrice or
mouthwash. The affected cells were removed by other
washes with saline solution, and the biofilms were disag-
gregated by ultrasonic homogenizer using a potency that
caused no damage to the structure of the microorganisms
(25%/30 s). Subsequently, the generated microbial suspen-
sion was diluted in saline solution and added in solid
medium to form colonies. For polymicrobial biofilms,
the suspensions were added in selective medium to de-
termine how much each specie was affected in the
mixed biofilm, both by the microbial interaction and by
the extract action. This analysis was performed by
counting of colony-forming units, being presented in
concentration per milliliter (CFU/mL).
Findings
R. officinalis L. extract provided a significant monomi-
crobial biofilms reduction after 5 min treatment, with
rates of 99.96 ± 0.07% for C. albicans; 67.84 ± 12.05% for
S. aureus; 77.64 ± 15.67% for E. faecalis; 79.32 ± 7.34%
for S. mutans; and 98.23 ± 2.17% for P. aeruginosa. Re-
garding the polymicrobial biofilms, the plant extract was
also effective due to a decreased CFU/mL concentration
observed in the treated groups. In the association of C.
albicans with S. aureus, the yeast was more affected (89
± 13.89%) compared to the bacterium (56.75 ± 22.58%).
In the biofilm of C. albicans with S. mutans was also ob-
served reductions of 92.04 ± 5.24% and 64.55 ± 15.12%,
respectively. On the other hand, the associations of C.
albicans (85.87 ± 17.48%) with E. faecalis (93.03 ± 2.44%)
and C. albicans (85.19 ± 10.48%) with P. aeruginosa
(83.33 ± 17.79%) significant differences were not found.
These results demonstrated the potential antibiofilm ef-
fect of R. officinalis L. extract on microorganisms that
may cause oral infections, as well as the possibility of
its insertion in oral hygiene materials to control bio-
films adhered to surfaces, such as teeth, oral mucosal,
prostheses, and orthodontic appliances.
Action mechanisms
Plant products have shown ability to act on biofilms ad-
hered to a surface [91]. In this way, these products can
inhibit the biofilm formation, prevent the planktonic
cells adhesion, and, consequently, block the microbial
colonization [101,102].
Plant extracts and phytocompounds can also impair the
microbial colonization. Microbes grown together with
plant products have shown less adhesion capacity, result-
ing in a biofilm formed by adhered cells that can be easily
removed [103].
A possible interaction target could be the bacterial
lipid bilayer. Carvacrol and thymol are chemically attracted
to the phospholipids of bacterial cytoplasmic membrane
and this interaction promotes loss of membrane integrity
and loss of cellular material, such as ions, adenosine tri-
phosphate (ATP), and genetic material [104,105]. The
hydrophobicity presented by some phytocompounds fa-
vors their diffusion through the polysaccharidic matrix
of the biofilm, promoting the destabilization of the mi-
crobial community [103].
Another proven mechanism is the interaction of plant
products with adhesive proteins located on the microbial
surface, preventing the attachment of new microorgan-
isms to the substrate or weaken the attachment of ad-
hered microorganisms [103].
In fungal species, da Silva Bomfim et al. [106] demon-
strated that R. officinalis L. essential oil affected the size
of Fusarium verticillioides microconidia, a fungus re-
sponsible for infecting grains such as corn and wheat.
This morphological alteration can impair the develop-
ment of the fungal biofilm. The mechanism involves tur-
gor pressure reduction on the fungal cell wall, as well as
changes in the cell surface caused by the need of os-
motic equilibrium restoration [107].
The antifungal effect of R. officinalis L. is result of its
interaction with the cell membrane and cell wall. The in-
tegrity of these structures is affected and all cytoplasmic
material is dispensed in the medium. This fact can be
verified by the presence of wrinkled cells in the fungal
biofilm [106,108].
Interruption of fungal cell growth, by the action of plant
products, can be related to the ergosterol biosynthesis in-
hibition, which is present in the cell membrane, as it is oc-
curred with antifungal drugs. In this sense, the membrane
integrity is affected and the functionality of its proteins is
also impaired, causing problems related to osmoregulatory
process, cell growth, and fungal proliferation [109].
Additionally, the antifungal activity of R. officinalis L. es-
sential oil has been related to the inhibition of C. albicans
germ tube formation, an important virulence factor used
for penetration and diffusion in organic tissues [110]. This
effect occurs due to oxidative stress generated by the plant
product, which triggers alterations in enzymatic activity
and potential of mitochondrial cell membrane. Thus, it is
possible inhibiting the germ-tube formation, yeast growth,
and promoting the fungal death [111].
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 9 of 22

Hepato-nephrotoxicity inhibition of the lead
Background
Lead (Pb) is a toxic heavy metal and harmful to living
things when mainly carried by food, water, and air. It
may present accentuated toxicity to the liver and kidneys
[112] as evidenced by a post-mortem analysis in individ-
uals intoxicated by Pb [113]. The contact with Pb is initi-
ated by its entry in the organism from drinking water
contaminated with the metal from the pipelines, canned
foods due to solder used in the cans, and ceramic enamels
[114]. Lead may provide a variety of disorders, including
hematological [115] and immunological changes [116],
cardiac [117], nervous [118], metabolic and reproductive
problems [119], and cancers [120]. However, ethylenedi-
aminetetraacetic acid (EDTA) calcium disodium salt, mag-
nesium dimercaptosuccinic acid (DMSA), D-penicillamine
(PCA), and dimercaprol (BAL) have been used to treated
Pb poisoning cases. These substances are chelating and
provide Pb reduction in the body [121]. However, these
medications can cause intoxication due to high dosage
or allergic reaction regards to the penicillin. The com-
mon side effects are: (i - EDTA) nephrotoxicity, head-
ache, fatigue, myalgia, thirst, fever, nausea and vomiting,
sneezing, nasal congestion, lacrimation, rashes, anemia,
and hypotension; (ii - DMSA) nausea, diarrhea, rashes,
transient elevation of the serum aminotransferase; (iii -
PCA) rheumatoid arthritis, urticaria, maculopapular re-
actions, lupus, pemphigoid, myasthenia gravis, renal
toxicity progressing to nephrotic syndrome, leukopenia,
thrombocytopenia, and aplastic anemia; and (iv - BAL)
rise in the blood pressure, tachycardia, vomiting, ab-
dominal pain, headache, burning sensation in mouth
and throat, lacrimation, blepharospasm, rhinorrhea, sweat-
ing, anxiety, fever, hemolytic anemia [122].
Methodology
Alternatively, R. officinalis L. ethanolic extract has been
evaluated as a protective option against the hepato-
nephrotoxic effect caused by the Pb [123]. In this study,
male albino rabbits received distilled water (control
group), R. officinalis L. extract or lead acetate (PbA)
for 30 days at 30 mg/kg. Also, another group of ani-
mals received plant extract for 30 days and then PbA
for the same period. Blood of sacrificed animals was
collected for analysis of total erythrocyte, packed cell
volume, hemoglobin, mean cell volume, mean corpus-
cular hemoglobin concentration, and total leukocyte,
granulocyte, lymphocyte, and monocyte. A biochem-
ical analysis was performed from the serum of the ani-
mals, verifying the presence of markers related to
damage in the liver and kidneys, as well as activities of
aspartate transaminase (AST), alanine aminotransfer-
ase (ALT), alkaline phosphatase (ALP), from liver, and
levels of urea (ERU) and creatinine (CRE), from kidneys.
The activity of catalase (CAT), superoxide dismutase
(SOD), and malondialdehyde (MDA) were quantified. Be-
sides, lipid peroxidation (LIP), glycogen (GLY), and tissues
protein (TSP) levels were checked. Histopathological and
histochemical analyses were performed by hematoxylin/
eosin staining and mercury bromophenol blue method,
respectively.
Findings
Animals exposed to the PbA showed significantly reduce
of activity and body weight. Absolute weight of liver and
kidneys also decreased to 42 and 62%, respectively. On
the other hand, the treatment with R. officinalis L. ex-
tract previously promoted normalization of the absolute
weight of liver (66%) and kidneys (80%). These data indi-
cated the protective effect of the plant extract against
the damages caused by Pb, regarding the changes in the
mass of the organs. The animals exposed for 30 days to
PbA presented a decreased in total erythrocyte, packed
cell volume, hemoglobin, mean cell volume and mean
corpuscular hemoglobin concentration. The number of
total leukocytes (neutrophils and monocytes) was in-
creased; however, the levels of lymphocyte and eosino-
phil were dramatically decreased. In rabbits pretreated
with plant extract, the cell concentration was similar to
the control group, demonstrating that the R. officinalis
L. extract had a protective effect, even with prolonged
exposure to the Pb. The production of markers related
to hepatic and renal damages was higher in animals ex-
posed to the PbA. The activity of AST (173%), ALT
(259%), ALP (162%) and the levels of ERU (161%) and
CRE (153%) were significantly increased. However, in ani-
mals pretreated with extract, lower concentrations of AST
(168%), ALT (129%), ALP (136%), ERU (121%) and CRE
(112%) were observed. Thus, the potential of R. officinalis
L. extract to protect the organisms against the harmful ef-
fects of the Pb was verified by biochemical tests. In ani-
mals exposed to PbA, the activity of antioxidant enzymes
was significantly reduced, including CAT (52%) and SOD
(47%), whereas MDA level (181%) was increased. In sam-
ples obtained from kidneys suspension, higher rates of
CAT (57%), SOD (62%), and MDA (375%) were found.
CAT (26%), SOD (45%) and MDA (63%) levels from the
liver, and CAT (16%), SOD (33%) and MDA (87%) levels
from kidneys were controlled only in animals pretreated
with plant extract. A significant glycogen reduction was
observed in the groups treated by PbA, both in the liver
(41%) and in the kidneys (21%). In contrast, the animals
treated with R. officinalis L. extract presented indexes of
20 and 7%, respectively. The tissue protein levels were sta-
tistically similar between the group exposed to PbA and
pretreated with extract, presenting reductions in both
cases. Histopathological analyses showed no alterations in
the liver of rabbits treated with R. officinalis L. extract or
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 10 of 22

distilled water (control), as well as in pretreated animals.
Histopathological changes were observed instead in ani-
mals exposed to the PbA, demonstrating necrotic areas.
Regarding the kidneys, abnormalities were not observed
in the control, pretreated and treated groups. In contrast,
severe tissue and pathological disorders were observed in
groups exposed to the PbA. High concentrations of neu-
tral mucopolysaccharides of hepatocytes and renal tubules
were demonstrated by histochemical analysis in rabbits
from control and treated groups. A carbohydrates reduc-
tion was observed in animals treated by the PbA, both in
their liver and kidneys; however, a higher concentration
was found in the animals preliminarily treated with R. offi-
cinalis L. extract. As for proteins, a reduction was ob-
served in the exposed groups to the PbA, both in the liver
and kidneys. Despite this, the protein content was not af-
fected in the group pretreated with the plant extract.
Hence, the protective effect of R. officinalis L. extract was
also histologically proven against hepato-nephrotoxicity of
the Pb.
Action mechanisms
Mohamed et al. [123] demonstrated the protective ability
of R. officinalis L. ethanolic extract in PbA-induced
hepato-nephrotoxicity. The study was carried out in rab-
bits and showed that PbA caused a significant hepatic
and renal dysfunction, compared to the animals not con-
taminated with PbA. These dysfunctions can be results
of changes in the cell membrane integrity, increased
ROS production, and lipid peroxidation [124]. In the
study by Mohamed et al. [123], rabbits pre-treated with
R. officinalis L. were protected against the harmful ef-
fects of PbA. The plant extract provided a reduction of
diffuse vacuolar cytoplasmic degeneration in hepatocytes
and renal tubules, besides decreased infiltration of lym-
phocytes in the liver and kidneys.
Additionally, in this study, loss of glycogen and liver
and renal proteins was identified after exposure to the
PbA. This fact occurred due to the interference of PbA
in absorption and metabolism of glucose, as well as in
induction of protein catabolism [125]. On the other
hand, R. officinalis L. inhibited the PbA action, and the
animals were not metabolically affected.
Regarding the hematological aspects, anemia was diag-
nosed in animals poisoned by PbA, probably caused by
enzymatic activity related to the metabolism of cell and
metal [126]. Inflammation induced by the intoxication
promoted elevated levels of leukocytes, neutrophils, and
monocytes. However, treatment with R. officinalis L.
provided low levels of anemia and normal levels of white
blood cells.
The protective hepato-nephro effect of R. officinalis L.
can be related to the interferences in oxidative stress
and lipid peroxidation caused by exposure to the PbA.
The plant extract restored the constitution of endogen-
ous antioxidants that were lost by the PbA intoxication,
besides regularizing the high levels of MDA. It has been
reported that R. officinalis L. can eliminate peroxyl radi-
cals and inhibit the formation of hydroxyl radicals [123].
Stress relief in situation of real danger
Background
In the face of imminent or fanciful danger, the organism
precedes these events and intensifies some chemical re-
actions, generating a series of physiological signals that
may affect the senses and some systems and cause symp-
toms, such as tachycardia, intense phobia, excessive per-
spiration, abdominal pain, and autonomic nervous system
dysfunction [127]. Thus, the anxiety is installed. For the
treatment, anxiolytics and antidepressants medications
have been used, including the benzodiazepines, which are
highly addictive and therefore should be consciously con-
sumed [128]. However, many of these medicines may
cause side effects such as hypotension, arrhythmias,
and anticholinergic effects [129]. Selective serotonin re-
uptake inhibitors (SSRIs) are medications used to treat
anxiety, being the most commonly prescribed antide-
pressants. Nevertheless, this drug may present side ef-
fects, including nausea, vomiting, insomnia, restlessness
and dysfunction [130]. Due to these undesirable results,
the use of plant medicines as adjuvant or primary treat-
ment has been considered to control psychiatric, and
neurological disorders [131].
Methodology
The antianxiety effect of R. officinalis L. hydroethanolic
extract was evaluated in rats submitted to a stressful
situation [132]. In this study, the animals received doses
of plant extract (100, 200 or 400 mg/kg) by intraperito-
neal injection. Rats from the control groups received
saline or diazepam (1 mg/kg). The effect of the prod-
ucts on anxiety in rats was evaluated by the elevated
plus maze device, which is used to generate and meas-
ure the anxiety, as well as to check the effect of anxio-
lytic medicines. It is a device composed of two
platforms (width: 10 cm; length: 40 cm) that cross each
other. One of the platforms is walled (high: 40 cm)
while the other has no wall. The center of the laby-
rinth has an area of 10 cm
2
, where the animal is placed
with its head facing the non-walled region. These plat-
forms are 50 cm above the ground. An illustration of
this device can be seen in Fig. 2.Thistestlastsfor5
min, and the animals are often stressed due to device
height and unprotected regions; thus, they tend to
seek shelter in walled areas. Hence, the anxiety may be
measured by the entries and permanence of the animal
in the protection-free area. Consequently, the absence
of anxiety is related to the ability to cope with these
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 11 of 22

challenges. The permanence time and the entries
number in each maze area are quantified separately.
The time spent and the entries percentage in unpro-
tected regions, as well as the locomotor activity,
should also be evaluated to measure the anxiety of the
animal. The entries number in any maze may measure
locomotor activity. This device was used in the study
by Abadi et al. [132], which rats were previously injected
with the products according to their experimental group
and the test was conducted after 45 min.
Findings
The permanence of the animals treated with R. officinalis
L. extract in the regions with no walls was increased, indi-
cating that these rats were less anxious and stressed. In
this evaluation, higher concentrations of the extract were
more effective and the dose of 400 mg/kg provided a simi-
lar effect to the diazepam. Additionally, the permanence
time in protected areas was reduced using the extract at
400 mg/kg, similar to the standard medicine. Regarding
the entries number in protected areas, a significant reduc-
tion was observed according to the dose, while in areas
with no walls the entries number was increased. By loco-
motor activity analysis, it was found that the animals of all
the groups presented similar behavior regarding the ex-
ploration of the maze. Based on these results, the antianxi-
ety potential of R. officinalis L. extract was effectively
demonstrated. The capacity this plant product to provide
stress relief in situations of real danger could be an alter-
native to the conventional medicines, which may lead to
various side effects and addiction.
Action mechanisms
The anxiolytic effect of R. officinalis L. can be attributed
to its potent antioxidant capacity. With this, the brain
can be protected by the many active molecules of the
plant against the damages caused by free radicals. Pos-
sible routes of action include oxidative stress reduction
and apoptosis inhibition that result in serotonergic neu-
rons protection and anxiety reduction. Besides the anti-
oxidant properties, R. officinalis L. has a significant anti-
inflammatory effect. Thus, the plant contributes to re-
duce the inflammatory mediator’s levels, control the pro-
tein denaturation, and decrease the dopaminergic and
serotonergic neuronal damages [132].
Anxiety has been treated with benzodiazepines, as
diazepam. Using interaction with brain receptors for
GABA neurotransmitter, diazepam can provide the
anxiolytic effect and also act as a sedative [133]. In the
study by Abadi et al. [132], administration of high
doses of R. officinalis L. hydroalcoholic extract pro-
vided a similar effect to this drug.
The anxiolytic effect of R. officinlais L. can occur due
to many phytocompounds in the plant. These molecules
may act throughout the central nervous system [132].
It has been known that R. officinlais L. is very rich in
flavonoids which work as ligands for central nervous
system receptors [134]. One of these flavonoids, is the
apigenin that can cross the blood-brain barrier and in-
crease the effect of GABA neurotransmitter on its re-
ceptor in the neuron. This is an important inhibitory
neurotransmitter of the central nervous system. Positively,
the plant product does not cause dependence, as the use
of benzodiazepines [135]. Another active flavonoid is the
Fig. 2 Elevated plus maze device, used to generate and measure the anxiety, as well as to check the effect of anxiolytic medicines. It is composed of
two platforms that cross each other. One of them is walled, while the other has no wall. The center of the labyrinth has an area of 10cm
2
, where the
animal is placed with its head facing the non-walled region. The test is conducted for 5min. Illustration based on real device
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 12 of 22

luteolin, capable of providing sedative and anxiolytic ef-
fects since it readily binds to the GABA receptors [136].
Human tumor cells proliferation inhibition
The nature of the tumors
Solid tumors consist of tumor cells and a characteris-
tic tumor vascularization, which is different from the
vascularization of healthy tissues. Thus, this micro-
environment is physiologically formed by high intersti-
tial fluid pressure (IFP), low oxygen tension, and low
extracellular pH. The regular balance of growth fac-
tors present in healthy tissues is totally unregulated in
a tumor tissue. This fact contributes to the develop-
ment of an abnormal vascularization that compro-
mises tissue structure and function. Therefore, the
nutrition and excretion of tumor tissue products are
compromised [137].
The high IFP in the tumor tissue is due to the vascu-
lar content accumulation caused by poor tumor
vascularization [138]. Thereby, factors such as de-
creased blood vessel activity and lymphatic, osmotic
pressure, and contractility of tumor stroma, cooperate
for increased IFP [138,139]. As a consequence, the
flow of cells with antitumor activity and therapeutic
substances are greatly impaired in tumor tissues [140].
It was verified a heterogeneous distribution of oxygen-
ation in the tumor tissue since some portions receive
low concentrations of oxygen and others are not
attended, due to the insufficient vascularization [141].
As for the extracellular pH, it was observed that tumor
tissues have acidic pH [142], due to the accumulation
of lactate, produced by the glucose metabolism, that
inside the tumor cell is found in high levels [143,144].
Therapeutic barriers to treat the cancer
Anticancer therapy can be compromised precisely by the
three factors cited above: high IFP, low oxygen tension
and low extracellular pH.
The high IFP in tumors compromises the delivery of
the antitumor agent, mainly antibodies and other pro-
teins, by decreasing the vascular flow, as well as its
transport from the circulation to the tumor. Patients
with lymphoma or melanoma have shown better results
with chemotherapy when decreased IFP occurs during
treatment [145].
Deficiencies in tissue oxygenation can inhibit the
therapeutic effect of radiation, since oxygen is a potent
radiosensitizer that contributes to tumor cell death
[146]. Also, hypoxia has also been reported as a problem
in the treatment with chemotherapeutic agents requiring
oxygen for maximum efficiency, such as mephalan, bleo-
mycin, and etoposide [147]. Besides, lack of oxygen com-
promises the cell division, thus, antiproliferative drugs
lose their effectiveness on tumor cells [148].
Acid extracellular pH can impair the delivery of many
chemotherapeutic agents [149]. The acidic condition in
the tumor tissue can affect many drugs at the molecular
level, preventing these agents from crossing the cell
membrane [150]. Additionally, some therapeutic mole-
cules are sequestered by acidic endosomes located inside
the tumor cell [151].
Breast adenocarcinoma (MCF-7) and cervical
adenocarcinoma (HeLa) [91]
Methodology
R. officinalis L. glycolic extract was added on the cells
previously cultured in microplates for 24 h. The analyses
were performed after exposure to the extract in different
concentrations (25, 50 and 100 mg/mL) for 5 min, using
the following assays: (i) MTT [3- (4,5-dimethylthiazo-
l-2-yl) -2,5-diphenyltetrazolium bromide], which mea-
sured the action of reductase enzymes in viable cells, by
the MTT degradation and formazan formation; (ii) neu-
tral red (NR), with the ability to impregnate lysosomes
in viable cells; (iii) crystal violet (CV), that can stain the
cellular genetic material; and (iv) genotoxicity, to verify
the micronuclei (MN) frequency, using a fluorescence
microscopy and DAPI dye that present affinity for the
genetic material. R. officinalis L. extract decreased the
viability of MCF-7 and HeLa, as evaluated by the MTT,
NR and CV assays.
Findings
At 100 mg/mL, a significant low cell viability was noted
by MTT, NR and CV. At 50 mg/mL, a reduction was
confirmed by NR and CV. On the other hand, at 25 mg/
mL, the cell viability was not significantly affected by
neither assay. Therefore, the R. officinalis extract in
higher concentrations interfered with the development
of tumor cells. Regarding the genotoxicity, the tested
concentrations induce no damage to the cellular genetic
material, since the MN frequency was significantly lower
(MCF-7) or similar to the control group (HeLa). This
can suggest that the R. officinalis L. extract protected the
cells against DNA damages. The damages could be more
harmful to these cells since they already present alter-
ations in their genetic material.
Epithelial colorectal adenocarcinoma (CaCo-2) and
histiocytic lymphoma cell line (U-937) [152]
Methodology
R. officinalis L. extract was obtained in ethyl acetate
(EAE) and methanol (MEE) at 0, 5, 10, 15, 20 and 25 μg/
mL. The effect of both extracts was evaluated on pri-
mary peripheral blood mononuclear cells (PBMC), a
non-tumoral line, for comparative purposes. Firstly, the
cells were cultured in microplates for 24 h, and then
were exposed to the extract for 72 h, checking the cell
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 13 of 22

viability every 24 h by Trypan blue exclusion test. Cell
cycle and apoptosis were evaluated by flow cytometry.
Early and late apoptosis were also measured by stain-
ing with propidium iodide DNA fluorochrome and
annexin test.
Findings
Both extracts presented a dose-dependent anti-proliferative
effect on CaCo-2 and U937. Besides, the extracts showed
better performance on tumor cells than on PBMC, after 48
h, since IC
50
was two and four-fold higher compared to
U937 and CaCo-2, respectively. This fact demonstrated the
selectivity of the extracts to act on tumor cells. By cell cycle
analysis, an increased of cell percentage in S phase was ob-
served. In contrast, a decreased cell population was noted
in G1 and G2/M phases. The EAE extract kept the cells in
the S phase (62%) longer, inhibiting their transition to G2/
M phase. On the other hand, MEE extract provided a de-
crease of CaCo-2 population in G2/M phase. Regarding the
apoptotic effect, CaCo-2 and U937 showed late apoptosis,
21.8 and 20.6%, respectively. These results can prove
that R. officinalis L. extracts inhibited the proliferation
of tumor cells.
Esophageal squamous cell carcinoma (KYSE30) and
gastric adenocarcinoma (AGS) [153]
Methodology
R. officinalis L aqueous extract was evaluated on adher-
ent cells in microplates after exposure for 24, 48 and 72
h. The analyses were performed by (i) MTT test; (ii) NR
assay; (iii) apoptosis with ethidium bromide/acridine or-
ange (EB/AO), evaluated by fluorescence microscopy,
which analyzed the condensed chromatin, apoptotic bod-
ies and necrotic cells; and (iv) cell cycle analysis (inter-
phase) by flow cytometry after DAPI staining.
Findings
R. officinalis L. extract affected the viability of KYSE30
and AGS after any exposure time. Regarding KYSE30,
IC
50
values were 600, 180, and 150 mg/mL, after 24, 48
and 72 h exposure, respectively, by MTT assay; and 860,
270, and 200 mg/mL, respectively, by NR assay. For
AGS, IC
50
values were remarkably lower, being 4.1, 1.8,
and 1.3 mg/mL, respectively, by MTT assay; and 4.4, 2.1,
and 1.1 mg/mL, respectively, by NR assay. Thus, the ex-
tract was more effective for the gastric adenocarcinoma
lineage. Besides, the cells showed fragmentation and
condensation of nucleus and chromatin, apoptotic bod-
ies formation and increased apoptotic cells amount,
proving that the plant extract induced the cell death.
These findings are in accordance with cell cycle results,
which demonstrated a higher percentage of cells in the
G1 phase (above 60%). However, this percentage was
below 30% in the S and G2/M phases, significant in
cases of cancers due theses phases are related to the
beginning and end of the DNA synthesis. Thereby, R.
officinalis L. extract acted as an antiproliferative agent,
interfering with the synthesis of defective genetic
material.
Lung carcinoma (A549) [154]
Methodology
The cells were treated with different concentrations (2.5,
5, 10, 25, 50, 100, 150 and 200 μg/mL) of R. officinalis L.
extract for 72 h. The antiproliferative effect of these con-
centrations was verified by (i) crystal violet test; (ii) clo-
nogenic assay, used to check the cell survival and ability
to form colonies; (iii) immunoblotting, used to quantify
proteins, such as PARP (related to apoptosis), Akt (re-
lated to cell proliferation), mTOR and p70S6K (both re-
lated to increased protein synthesis and cell survival).
Findings
R. officinalis L. extract presented IC
50
of 15.9 μg/mL.
Additionally, the extract at 2.5 μg/mL inhibited the col-
ony formation (39.3 ± 3.1% of control) and at 10 μg/mL
almost caused total elimination (1.2 ± 3.1% of control) as
seen by clonogenic assay. This fact demonstrated the po-
tential of the extract to control the stabilization and de-
velopment of tumor cells, essential factors for the tumor
growth in living beings. The PARP levels decreased to
50 μg/mL, thus the extract could contribute to improv-
ing the apoptosis process. Besides, R. officinalis L. ex-
tract inhibited the Akt phosphorylation, contributing to
the non-activation of this protein that is related to pro-
liferation and survival of A549 cells. In this way, the
most effective concentrations were 25 μg/mL (57 ± 5.04%
of control) and 50 μg/mL (36.1 ± 4.9% of control). The
Akt levels also decreased at 25 μg/mL (49.8 ± 5.3% of
control) and 50 μg/mL (32.4 ± 0.7% of control). There-
fore, R. officinalis L. extract could interfere with the Akt
signaling in these tumor cells. When Akt is activated,
the signaling of mTOR and p70S6K may occur and re-
sult in increased protein synthesis, proliferation, and cell
survival. However, the extract provided a significant in-
hibition of mTOR phosphorylation (53.3 ± 10.9% of con-
trol) and p70S6K (57.2 ± 14.8% of control) at 50 μg/mL.
Low levels of mTOR (84.5 ± 2.5% of control) and p70S6K
(83.3 ± 2.5% of control) were also observed. Based on
these results, R. officinalis L. extract inhibited the A549
proliferation, interfering in some mechanisms related to
colonization, proliferation, survival and apoptosis.
Action mechanisms
Cancer cells can survive and develop tumors higher than
non-tumor cells, even under chemo- and radiotherapy
conditions. Among the strategies used by cells, the cap-
acity to form new colonies is an important ability they
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 14 of 22

present [154]. On the other hand, these authors proved
that R. officinalis L. extract could inhibit the formation
of new colonies of lung cancer cells (A549).
The tumor cells proliferation occurs on the oxidative
stress influence, which exerts on the cells a force for them
to survive to this adversity. Therefore, this situation acti-
vates the redox signaling and, consequently, activator pro-
tein (AP-1) and NF-κB are also activated. Subsequently,
tumor suppressor genes inhibition can also be observed
[155]. In contrast, the antitumor activity of R. officinalis L.
has been attributed to the antioxidant effect that the plant
presents, such as free radicals elimination and lipid perox-
idation control [156,157]. This fact has been proven on
cancerous lineages such as MCF-7 and colorectal adeno-
carcinoma cells (HT-29) [158,159].
The cytotoxicity of R. officinalis L. observed on tumor
cells can be related to the interference in the cell cycle
and also to apoptosis induction. R. officinalis L. metha-
nolic and ethyl acetate extracts impaired distribution of
U937 cell cycle in S phase, and provided a decrease in
the G1 and G2/M phases. In addition, the methanolic
fraction of the extract inhibited the CaCo-2 growth, with
a decrease in the G2/M phase [152].
In response to the action of a therapeutic agent, the
tumor cell can release ROS to provide the necessary oxida-
tive stress for its proliferation, using the mitochondrial
pathway [160,161]. However, rosmanol, a phytocompound
from R. officinalis L., caused apoptosis in colorectal adeno-
carcinoma cells (COLO 205), increasing the levels of
apoptosis-inducing factor (AIF) and cytochrome c [162].
Other phytocompounds from R. officinalis L. have been
cited as responsible for antiproliferative activity against
cancer cells, as the ursolic acid that can act on the NF-κB
pathway and provide NF-κB phosphorylation repressors
inhibition. Thus, this phytocompound can attenuate the
action of agents involved in oncogenesis, such as COX-2,
MMP-9, cyclin D1, C-Jun, and C-fos. The antioxidant po-
tential of ursolic acid has also been observed [153]. Be-
sides, carnosol has shown a blocking effect on NF-κB
[163], and carsonic acid has neutralized ROS and, conse-
quently, protecting cell membranes against lipid peroxida-
tion [153].
There is a class of proteins that after activation by
cleavage acts on the DNA repair or even lead the cell to
apoptosis, in case of impossible repair of the genetic ma-
terial. These enzymes are called poly ADP ribose poly-
merase (PARP), and were activated by DNA breaking
caused by ROS or other reactive species [164,165]. In
the study conducted by Moore et al. [154] increased
PARP cleavage was observed in A549 cells by exposure
to the R. officinalis L. extract, indicating an induction to
the apoptosis of these cancer cells. These authors also
proved that the plant extract can control the Akt phos-
phorylation, an important enzyme responsible for the
regulation of metabolism, apoptosis, and cell prolifera-
tion. Blocking of this pathway (P13K/Akt) can result in
improvements in the cancer treatment by chemo- or
radiotherapeutic agents [166]. Besides, Moore et al.
[154] have also shown that the extract can inhibit the ac-
tivation of mTOR and p70S6K, mammalian targets of
rapamycin that are cancer signaling proteins. Probably,
this occurs due to the ability of R. officinalis L. extract in
interfering with protein synthesis by DDIT4 gene induc-
tion, which is capable of inhibiting the synthesis of
mTOR and p70S6k [167].
Final considerations and conclusions
In this Review, some pharmacological effects of products
from R. officinalis L. were shown. These effects were
widely demonstrated on diverse types of disorders in-
cluding (a) cardiac remodeling after myocardial infarc-
tion [47]; (b) body weight and dyslipidemia [60]; (c)
cerebral ischemia [71]; (d) pain [77]; (e) infections [91];
(f) hepato-nephrotoxicity by lead [123]; (g) stress and
anxiety [132]; and (h) tumor cells proliferation [91,
152–154]. Thus, with this study, it was possible to verify
the benefits of R. officinalis L. on specific health problems
that can affect many people around the world.
Supplementation with fresh leaves from R. officinalis
L. provided better survival rates in infarcted animals, as
well as improving diastolic function, cardiac muscle
hypertrophy, and heart functions and morphology, com-
pared to the animals receiving only conventional treat-
ment. Thereby, it was demonstrated that the use of this
plant could be a complementary therapy to the usual
procedure of cardiac remodeling after infarction [47].
The use of some drugs, like antibiotics, can cause in-
creased lipids and fats in the blood, known as dyslipid-
emia. Accumulation of these molecules in the vessels
can promote cardiovascular and cerebrovascular dis-
eases. However, administration of R. officinalis L. aque-
ous extract to the animals with gentamicin-induced
dyslipidemia provided weight gain inhibition, caused by
fat accumulation due to treatment with the antibiotic.
Other positive aspects were the liver weight reduction of
these animals and organ repair to the trauma generated
by the medicament. Thus, the plant extract could be a
strong candidate to control dyslipidemia and, conse-
quently, reduce the chances of manifestations, such as
heart attack, angina, and stroke, which have affected sev-
eral individuals in all continents [60].
Stroke is precisely caused by disruption of blood flow
in the brain, either by a clogging (ischemia) or by a rup-
tured vessel (hemorrhagic stroke). However, both cause
irreversible damage to the affected region that will com-
promise the individual’s performance in some way. R.
officinalis L. hydroethanolic extract has been shown a
neuroprotective effect in animals with artificially induced
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 15 of 22

cerebral ischemia and previously treated with the plant
product, presenting reduced neurological deficit. In
addition, the weight of the animals and their lipid in-
dexes in the circulation were controlled by the use of R.
officinalis L. extract. These findings demonstrated a
prophylactic effect of R. officinalis L. on an essential
clinical manifestation caused by disruption of blood cir-
culation in the brain [71].
Some medications to treat pain can cause side effects,
such as cardiovascular, renal, and gastric problems. Thus,
the effectiveness of R. officinalis L. ethanol extract and its
phytocompounds ursolic acid and oleanolic acid was ob-
served in animals previously treated with the plant ex-
tracts, and with formalin-induced nociception. Plant
products administrated in association with ketorolac
improved the antinociceptive effect. Thus, this study
showed that products from R. officinalis L. did not
affect the efficiency of an allopathic drug, in contrast,
they potentialized the pain control, in a complementary
manner [77].
Another subject of relevance to the medical commu-
nity is the emergence of opportunistic microorganisms
and resistant to the available antimicrobial treatments.
Because of this, R. officinalis L. glycolic extract was used
in alternative to those therapeutic agents to control the
development of mono- and polymicrobial biofilms.
Thus, significant reductions of microbial communities
were observed after the plant extract application. There-
fore, the plant product could be a potential alternative
therapeutic agent to eliminate microorganisms and, con-
sequently, inhibit the development of infections that can
culminate in a fatality [91].
Lead intoxication is a public health problem in many
countries due to the aspects of subsistence of their com-
munities. Several disorders can be reported, and the
treatment can also be harmful in the same way as heavy
metal intoxication. Therefore, R. officinalis L. ethanolic
extract has been shown as a protector to the liver and
kidneys against the toxic effect promoted by the Pb. Pro-
longed use of the extract has inhibited the degrading ac-
tion of Pb, such as loss of body weight and weight
reduction of liver and kidneys, blood cells reduction, in-
crease in the circulation of markers related to the liver
and kidney damage, and an increase of necrotic areas in
these organs. In this way, the use of R. officinalis L.
could be a way for these communities to prevent the
harmful effects of Pb [123].
R. officinalis L. has also been tested for anxiety, as an
alternative to the available antianxiety and antidepressants,
which can present side effects, including hypotension, ar-
rhythmias, and addiction. For this, animals were submitted
to a stressful situation; however, they previously were
treated with R. officinalis L. hydroethanolic extract. It
was noticed a significant anxiety control similar to the
diazepam. Many anxiolytic medications are useful, but
cases of addiction have been reported. Thus, R. officinalis
L. could be an alternative for these cases, since its prophy-
lactic effect against anxiety has been proven [132].
Another malignancy that has advanced in all countries
and caused the death of thousands of people is cancer.
Regarding the therapy, many agents anticancer have
been studied for the treatment of the disease, as antitu-
mor and antiproliferative drugs. One of the problems in-
volved would be the drugs reaching the target since the
tumor microenvironment limits the diffusion of these
drugs. Another considered aspect is the side effect of an-
titumor therapies. Therefore, the development of alter-
native methods less invasive with fewer side effects to
the patients has been discussed. An example of this is
the experiments with plant products, such as antioxidant
molecules and extracts. Products from R. officinalis L.
have been evaluated and demonstrated effective antipro-
liferative action on several types of tumor cells. These
products have provided interference in the cell cycle, as
well as promoting apoptosis, in order to inhibit the pro-
liferation and colonization of malignant cells. Besides,
these products have also been biocompatible for other
cell types. Thus, the results have been promising and
could be effective against the tumor cells, with preserva-
tion of the healthy cells and the minimum of damage for
the organism of the patient [91,152–154].
Therefore, many research groups around the world
have been engaging in the development of alternative
and biocompatible products to treat the most diverse
physiological disorders that affect humans. Conventional
medications are effective; however, they can offer several
side effects, including severe morbidities. Phytotherapy
medicines, those that are produced from plant products,
such as phytocompounds, extracts, essential oils, and
tinctures, have been used as alternative or complemen-
tary medicines, due to scientific evidence of their benefi-
cial effects.
In this Review, reports on R. officinalis L. benefits
were presented to show that a plant product may con-
trol physiological disorders similar to or superior to the
usual medications. Another point to consider is the
demonstration of new treatment forms and pharmaco-
logical strategies that could be developed to reach as
many people as possible in all Continents.
Abbreviations
5-HT1A: 5-hydroxytryptamine receptors; A549: Lung carcinoma; AGS: Gastric
adenocarcinoma; AIF: Apoptosis-inducing factor; Akt: Protein related to cell
proliferation; ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AP-
1: Activator protein; AST: Aspartate transaminase; ATP: Adenosine triphosphate;
BAL: Dimercaprol; Caco-2: Epithelial colorectal adenocarcinoma; CAT: Catalase;
CFU/mL: Colony-forming units per milliliter; cGMP: Cyclic guanosine
monophosphate; COLO 205: Colorectal adenocarcinoma cells; COX-
2: Cyclooxygenase-2; CRE: Creatinine; CV: Crystal violet; DMSA: Mgnesium
dimercaptosuccinic acid; EAE: Ethyl acetate; EB/AO: Ethidium bromide/
acridine Orange; EDTA: Ethylenediaminetetraacetic acid; ERU: Urea;
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 16 of 22

GABA: γ-aminobutyric acid; GLY: Glycogen; HDL-c: High density lipoprotein;
HeLa: Cervical adenocarcinoma; HMG-CO: 3-hydroxy-3-methylglutaryl
coenzyme A reductase; IC
50
: Half maximal inhibitory concentration; IFN-
γ: Interferon gamma; IFP: Interstitial fluid pressure; IL-10: Interleukin 10; IL-
1β: Interleukin 1 beta; IL-6: Interleukin 6; iNOS: Nitric oxide synthase;
KYSE30: Esophageal squamous cell carcinoma; LDL-c: Low density
lipoprotein; LIP: Lipid peroxidation; MCF-7: Breast adenocarcinoma;
MDA: Malondialdehyde; MEE: Methanol; MMP: Matrix metalloproteinases;
MN: Micronuclei; mTOR: Protein related to increased protein synthesis and
cell survival; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; NaCl: Sodium chloride; NADH: Nicotinamide adenine dinucleotide
hydride; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B
cells; NO: Nitric oxide; NR: Neutral red; Nrf2: Nuclear factor (erythroid-derived 2)-
like 2; NSAIDs: Non-steroidal anti-inflammatory drugs; p70S6K: Protein related to
increased protein synthesis and cell survival; PARP: Poly ADP ribose polymerase;
Pb: Lead; PbA: Lead acetate; PBMC: Peripheral blood mononuclear cells; PCA: D-
penicillamine; ROS: Reactive oxygen species; SOD: Superoxide dismutase;
SSRIs: Selective serotonin reuptake inhibitors; TC: Total cholesterol;
TG: Triglyceride; TIMP-1: Tissue inhibitor of metalloproteinases 1; TNF-
α: Tumor necrosis factor alpha; TPA: Tissue plasminogen activator;
TrpV: Transient receptor potential cation channel subfamily V member 1;
TSP: Tissues protein; U-937: Histiocytic lymphoma cell line
Acknowledgements
Not applicable.
Funding
None.
Availability of data and materials
Not applicable.
Authors’contributions
JRO, conception and drafting of the manuscript; SEAC, review of the manuscript;
LDO, review of the manuscript. All authors read and approved the final
manuscript.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in published
maps and institutional affiliations.
Author details
1
Departamento de Biociências e Diagnóstico Bucal, Instituto de Ciência e
Tecnologia, Universidade Estadual Paulista (UNESP), Av. Engenheiro Francisco
José Longo, 777 –Jardim São Dimas, São José dos Campos, SP CEP
12245-000, Brazil.
2
Department of Restorative Dental Sciences, University of
Florida, College of Dentistry, Gainesville, FL 32610, USA.
Received: 26 July 2018 Accepted: 2 January 2019
References
1. González-Trujano ME, Peña EI, Martínez AL, Moreno J, Guevara-Fefer P,
Déciga-Campos M, et al. Evaluation of the antinociceptive effect of
Rosmarinus officinalis L. using three different experimental models in
rodents. J Ethnopharmacol. 2007;111:476–82.
2. Pérez-Fons L, Garzón MT, Micol V. Relationship between the antioxidant
capacity and effect of rosemary (Rosmarinus officinalis L.) polyphenols on
membrane phospholipid order. J Agric Food Chem. 2010;58:161–71.
3. Brewer MS. Natural antioxidants: sources, compounds, mechanisms of
action, and potential applications. Compr Rev Food Sci Food Saf. 2011;10:
221–47.
4. RaškovićA, MilanovićI, PavlovićN, ĆebovićT, VukmirovićS, Mikov M.
Antioxidant activity of rosemary (Rosmarinus officinalis L.) essential oil and its
hepatoprotective potential. BMC Complement Altern Med. 2014;14:225.
5. Martínez AL, González-Trujano ME, Pellicer F, López-Muñoz FJ, Navarrete A.
Antinociceptive effect and GC/MS analysis of Rosmarinus officinalis L.
essential oil from its aerial parts. Planta Med. 2009;75:508–11.
6. Borges RS, Ortiz BLS, Pereira ACM, Keita H, Carvalho JCT. Rosmarinus officinalis
essential oil: A review of its phytochemistry, anti-inflammatory activity, and
mechanisms of action involved. J Ethnopharmacol. 2019;229:29–45.
7. Einbond LS, Wu HA, Kashiwazaki R, He K, Roller M, Su T, et al. Carnosic acid
inhibits the growth of ER-negative human breast cancer cells and
synergizes with curcumin. Fitoterapia. 2012;83:1160–8.
8. Gonçalves GA, Corrêa RCG, Barros L, Dias MI, Calhelha RC, Correa VG, et al.
Effects of in vitro gastrointestinal digestion and colonic fermentation on a
rosemary (Rosmarinus officinalis L) extract rich in rosmarinic acid. Food
Chem. 2019;271:393–400.
9. al-Sereiti MR, Abu-Amer KM, Sen P. Pharmacology of rosemary
(Rosmarinus officinalis Linn.) and its therapeutic potentials. Indian J
Exp Biol. 1999;37:124–30.
10. Hsieh CL, Peng CH, Chyau CC, Lin YC, Wang HE, Peng RY. Low-density
lipoprotein, collagen, and thrombin models reveal that Rosemarinus
officinalis L. exhibits potent antiglycative effects. J Agric Food Chem.
2007;55:2884–91.
11. Scheckel KA, Degner SC, Romagnolo DF. Rosmarinic acid antagonizes
activator protein-1-dependent activation of cyclooxygenase-2 expression in
human cancer and nonmalignant cell lines. J Nutr. 2008;138:2098–105.
12. Karthik D, Viswanathan P, Anuradha CV. Administration of rosmarinic acid
reduces cardiopathology and blood pressure through inhibition of p22phox
NADPH oxidase in fructose-fed hypertensive rats. J Cardiovasc Pharmacol.
2011;58:514–21.
13. Fernández LF, Palomino OM, Frutos G. Effectiveness of Rosmarinus officinalis
essential oil as antihypotensive agent in primary hypotensive patients and its
influence on health-related quality of life. J Ethnopharmacol. 2014;151:509–16.
14. Amaral GP, de Carvalho NR, Barcelos RP, Dobrachinski F, Portella Rde L, da
Silva MH, et al. Protective action of ethanolic extract of Rosmarinus officinalis
L. in gastric ulcer prevention induced by ethanol in rats. Food Chem
Toxicol. 2013;55:48–55.
15. Posadas SJ, Caz V, Largo C, De la Gándara B, Matallanas B, Reglero G, et al.
Protective effect of supercritical fluid rosemary extract, Rosmarinus officinalis,
on antioxidants of major organs of aged rats. Exp Gerontol. 2009;44:383–9.
16. Kayashima T, Matsubara K. Antiangiogenic effect of carnosic acid and
carnosol, neuroprotective compounds in rosemary leaves. Biosci Biotechnol
Biochem. 2012;76:115–9.
17. Sinkovic A, Suran D, Lokar L, Fliser E, Skerget M, Novak Z, et al. Rosemary
extracts improve flow-mediated dilatation of the brachial artery and plasma
PAI-1 activity in healthy young volunteers. Phytother Res. 2011;25:402–7.
18. Cheung S, Tai J. Anti-proliferative and antioxidant properties of rosemary
Rosmarinus officinalis. Oncol Rep. 2007;17:1525–31.
19. Yesil-Celiktas O, Sevimli C, Bedir E, Vardar-Sukan F. Inhibitory effects of
rosemary extracts, carnosic acid and rosmarinic acid on the growth of
various human cancer cell lines. Plant Foods Hum Nutr. 2010;65:158–63.
20. Menghini L, Genovese S, Epifano F, Tirillini B, Ferrante C, Leporini L.
Antiproliferative, protective and antioxidant effects of artichoke, dandelion,
turmeric and rosemary extracts and their formulation. Int J Immunopathol
Pharmacol. 2010;23(2):601–10.
21. Kontogianni VG, Tomic G, Nikolic I, Nerantzaki AA, Sayyad N, Stosic-Grujicic
S, et al. Phytochemical profile of Rosmarinus officinalis and Salvia officinalis
extracts and correlation to their antioxidant and anti-proliferative activity.
Food Chem. 2013;136:120–9.
22. Nolkemper S, Reichling J, Stintzing FC, Carle R, Schnitzler P. Antiviral effect
of aqueous extracts from species of the Lamiaceae family against herpes
simplex virus type 1 and type 2 in vitro. Planta Med. 2006;72:1378–82.
23. Bernardes WA, Lucarini R, Tozatti MG, Flauzino LG, Souza MG, Turatti IC, et
al. Antibacterial activity of the essential oil from Rosmarinus officinalis and its
major components against oral pathogens. Z Naturforsch C. 2010;65:588–93.
24. Ramadan KS, Khalil OA, Danial EN, Alnahdi HS, Ayaz NO. Hypoglycemic and
hepatoprotective activity of Rosmarinus officinalis extract in diabetic rats. J
Physiol Biochem. 2013;69:779–83.
25. El Saied Azab A. Nephro-protective effects of curcumin, rosemary, and
propolis against gentamicin-induced toxicity in Guinea pigs: morphological
and biochemical study. Am J Clin Exp Med. 2014;2:28.
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 17 of 22

26. Del Baño MJ, Castillo J, Benavente-García O, Lorente J, Martín-Gil R, Acevedo
C, et al. Radioprotective-antimutagenic effects of rosemary phenolics
against chromosomal damage induced in human lymphocytes by gamma-
rays. J Agric Food Chem. 2006;54:2064–8.
27. al-Hader AA, Hasan ZA, Aqel MB. Hyperglycemic and insulin release
inhibitory effects of Rosmarinus officinalis. J Ethnopharmacol. 1994;43:
217–21.
28. Tabassum N, Hamdani M. Plants used to treat skin diseases. Pharmacogn
Rev. 2014;8:52–60.
29. Machado DG, Cunha MP, Neis VB, Balen GO, Colla AR, Grando J, et al.
Rosmarinus officinalis L. hydroalcoholic extract, similar to fluoxetine, reverses
depressive-like behavior without altering learning deficit in olfactory
bulbectomized mice. J Ethnopharmacol. 2012;143:158–69.
30. Vongsak B, Sithisarn P, Mangmool S, Thongpraditchote S, Wongkrajang Y,
Gritsanapana W. Maximizing total phenolics, total flavonoids contents and
antioxidant activity of Moringa oleifera leaf extract by the appropriate
extraction method. Ind Crop Prod. 2013;44:566–71.
31. Azwanida NN. A review on the extraction methods use in medicinal plants,
principle, strength and limitation. Med Aromat Plants. 2015;4:1–6.
32. Borhan MZ, Ahmad R, Mohd RM, Abdullah S. Impact of nano powders on
extraction yield of Centella asiatica. Adv Mater Res. 2013;667:246–50.
33. Arya V, Thakur NM, Kashyap C. Preliminary phytochemical analysis of the
extracts of Psidium leaves. J Pharmacogn Phytochem. 2012;1:1–5.
34. Al-Mansoub MA, Asmawi MZ, Murugaiyah V. Effect of extraction solvents
and plant parts used on the antihyperlipidemic and antioxidant effects of
Garcinia atroviridis: a comparative study. J Sci Food Agric. 2014;94:1552–8.
35. Yung OH, Maskat MY, Wan Mustapha WA. Effect of extraction on
polyphenol content, antioxidant activity and pH in pegaga (Centella
asiatica). Sains Malaysiana. 2010;39:747–52.
36. Puttarak P, Panichayupakaranant P. A new method for preparing pentacyclic
triterpene rich Centella asiatica extracts. Nat Prod Res. 2013;27:684–6.
37. Hori M, Nishida K. Oxidative stress and left ventricular remodelling after
myocardial infarction. Cardiovasc Res. 2009;81:457–64.
38. Zornoff LA, Paiva SA, Minicucci MF, Spadaro J. Experimental myocardium
infarction in rats: analysis of the model. Arq Bras Cardiol. 2009;93:434–40
426-32.
39. Cohn JN, Archibald DG, Ziesche S, Franciosa JA, Harston WE, Tristani FE, et
al. Effect of vasodilator therapy on mortality in chronic congestive heart
failure: results of a veterans administration cooperative study. N Engl J Med.
1986;314:1547–52.
40. Goldstein RE, Boccuzzi SJ, Cruess D, Nattel S. Diltiazem increases late-onset
congestive heart failure in postinfarction patients with early reduction in
ejection fraction. The adverse experience committee; and the multicenter
diltiazem Postinfarction research group. Circulation. 1991;83:52–60.
41. Cohn JN, Ziesche S, Smith R, Anand I, Dunkman WB, Loeb H, et al. Effect of
the calcium antagonist felodipine as supplementary vasodilator therapy
in patients with chronic heart failure treated with enalapril: V-HeFT III.
Vasodilator-heart failure trial (V-HeFT) study group. Circulation. 1997;96:
856–63.
42. Packer M, Carver JR, Rodeheffer RJ, Ivanhoe RJ, DiBianco R, Zeldis SM, et al.
Effect of oral milrinone on mortality in severe chronic heart failure. The
PROMISE study research group. N Engl J Med. 1991;325:1468–75.
43. Lubsen J, Just H, Hjalmarsson AC, La Framboise D, Remme WJ, Heinrich-
Nols J, et al. Effect of pimobendan on exercise capacity in patients with
heart failure: main results from the Pimobendan in congestive heart failure
(PICO) trial. Heart. 1996;76:223–31.
44. Hampton JR, van Veldhuisen DJ, Kleber FX, Cowley AJ, Ardia A, Block P, et
al. Randomised study of effect of ibopamine on survival in patients with
advanced severe heart failure. Second Prospective Randomised Study of
Ibopamine on Mortality and Efficacy (PRIME II) Investigators. Lancet. 1997;
349:971–7.
45. Cohn JN, Goldstein SO, Greenberg BH, Lorell BH, Bourge RC, Jaski BE, et al.
A dose-dependent increase in mortality with vesnarinone among patients
with severe heart failure. Vesnarinone trial investigators. N Engl J Med. 1998;
339:1810–6.
46. Genena AK, Hense H, Junior AS, Souza SM. Rosemary (Rosmarinus officinalis) -
a study of the composition, antioxidant and antimicrobial activities of
extracts obtained with supercritical carbon dioxide. Ciênc Tecnol Aliment.
2008;28:463–9.
47. Murino Rafacho BP, Portugal Dos Santos P, Gonçalves AF, AAH F, Okoshi K,
Chiuso-Minicucci F, et al. Rosemary supplementation (Rosmarinus oficinallis
L.) attenuates cardiac remodeling after myocardial infarction in rats. PLoS
One. 2017;12:e0177521.
48. Cohn JN, Ferrari R, Sharpe N. Cardiac remodeling-concepts and clinical
implications: a consensus paper from an international forum on cardiac
remodeling. Behalf of an international forum on cardiac remodeling. J Am
Coll Cardiol. 2000;35:569–82.
49. Madamanchi NR, Runge MS. Redox signaling in cardiovascular health and
disease. Free Radic Biol Med. 2013;61:473–501.
50. van Deel ED, Lu Z, Xu X, Zhu G, Hu X, Oury TD, et al. Extracellular superoxide
dismutase protects the heart against oxidative stress and hypertrophy after
myocardial infarction. Free Radic Biol Med. 2008;44:1305–13.
51. Liu T, Chen L, Kim E, Tran D, Phinney BS, Knowlton AA. Mitochondrial
proteome remodeling in ischemic heart failure. Life Sci. 2014;101:27–36.
52. Chohan M, Naughton DP, Opara EI. Determination of superoxide dismutase
mimetic activity in common culinary herbs. Springerplus. 2014;3:578.
53. Sun GB, Sun X, Wang M, Ye JX, Si JY, Xu HB, et al. Oxidative stress
suppression by luteolin-induced heme oxygenase-1 expression. Toxicol
Appl Pharmacol. 2012;265:229–40.
54. Sahu BD, Putcha UK, Kuncha M, Rachamalla SS, Sistla R. Carnosic acid
promotes myocardial antioxidant response and prevents isoproterenol-
induced myocardial oxidative stress and apoptosis in mice. Mol Cell
Biochem. 2014;394:163–76.
55. McBride P. Triglycerides and risk for coronary artery disease. Curr Atheroscler
Rep. 2008;10:386–90.
56. Naerr GW, Rein P, Saely CH, Drexel H. Effects of synthetic and biological
disease modifying antirheumatic drugs on lipid and lipoprotein parameters
in patients with rheumatoid arthritis. Vasc Pharmacol. 2016;81:22–30.
57. Rojo LE, Gaspar PA, Silva H, Risco L, Arena P, Cubillos-Robles K, et al.
Metabolic syndrome and obesity among users of second generation
antipsychotics: a global challenge for modern psychopharmacology.
Pharmacol Res. 2015;101:74–85.
58. Nsagha DS, Weledji EP, Assob NJ, Njunda LA, Tanue EA, Kibu OD, et al.
Highly active antiretroviral therapy and dyslipidemia in people living with
HIV/AIDS in Fako Division, South West Region of Cameroon. BMC
Cardiovasc Disord. 2015;15:95.
59. Ahmadvand H, Ghasemi Dehnoo M, Dehghani A, Bagheri S, Cheraghi RA.
Serum paraoxonase 1 status and its association with atherogenic indexes in
gentamicin-induced nephrotoxicity in rats treated with coenzyme Q10. Ren
Fail. 2014;36:413–8.
60. Hegazy AM, Abdel-Azeem AS, Zeidan HM, Ibrahim KS, Sayed EE.
Hypolipidemic and hepatoprotective activities of rosemary and thyme in
gentamicin-treated rats. Hum Exp Toxicol. 2018;37:420–30.
61. Wojdyloa A, Oszmian’skia J, Czemerys R. Antioxidant activity and phenolic
compounds in 32 selected herbs. Food Chem. 2007;105:940–9.
62. Abbas MT, Hashimzyni MM, Al-Kadhi NA. Effect of garlic oil on gentamicin
induced hapatorenal toxicity in rats. J Kerbala Univ. 2013;11:109–17.
63. Lin CY, Chen JH, Fu RH, Tsai CW. Induction of pi form of glutathione S.
transferase by carnosic acid is mediated though P13k/Akt/NFKB pathway
and protects against neurotoxicity. Chem Res Toxicol. 2014;27:1958–66.
64. Yokozawa T, Nakagawa T, Kitani K. Antioxidative activity of green tea
polyphenol in cholesterol-fed rats. J Agric Food Chem. 2002;50:3549–52.
65. Raza SS, Khan MM, Ahmad A, Ashafaq M, Islam F, Wagner AP, et al.
Neuroprotective effect of naringenin is mediated through suppression of
NF-κB signaling pathway in experimental stroke. Neuroscience. 2013;230:
157–71.
66. Liu Z, Liu Q, Cai H, Xu C, Liu G, Li Z. Calcitonin gene-related peptide
prevents blood-brain barrier injury and brain edema induced by focal
cerebral ischemia reperfusion. Regul Pept. 2011;171:19–25.
67. Huang P, Zhou CM, Qin-Hu LYY, Hu BH, Chang X, et al. Cerebralcare
granule® attenuates blood-brain barrier disruption after middle cerebral
artery occlusion in rats. Exp Neurol. 2012;237:453–63.
68. Liu F, Akella P, Benashski SE, Xu Y, McCullough LD. Expression of Na-K-cl
cotransporter and edema formation are age dependent after ischemic
stroke. Exp Neurol. 2010;224:356–61.
69. Heo JH, Han SW, Lee SK. Free radicals as triggers of brain edema formation
after stroke. Free Radic Biol Med. 2005;39:51–70.
70. Abu Fanne R, Nassar T, Yarovoi S, Rayan A, Lamensdorf I, Karakoveski M, et
al. Blood-brain barrier permeability and tPA-mediated neurotoxicity.
Neuropharmacology. 2010;58:972–80.
71. Seyedemadi P, Rahnema M, Bigdeli MR, Oryan S, Rafati H. The
neuroprotective effect of rosemary (Rosmarinus officinalis L.) hydro-alcoholic
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 18 of 22

extract on cerebral ischemic tolerance in experimental stroke. Iran J Pharm
Res. 2016;15:875–83.
72. Rekik I, Allassonniere S, Carpenter TK, Wardlaw JM. Medical image analysis
methods in MR/CTimaged acute-subacute ischemic stroke lesion:
segmentation, prediction and insights into dynamic evolution simulation
models. A critical appraisal. Neuroimage Clin. 2012;1:164–78.
73. Raza SS, Khan MM, Ahmad A, Ashafaq M, Islam F, Wagner AP, et al.
Neuroprotective effect of naringenin is mediated through suppression of NF-
?B signaling pathway in experimental stroke. Neuroscience. 2013;230:157–71.
74. Huang P, Zhou CM, Hu Q, Liu YY, Hu BH, Chang X, et al. Cerebral care
granule attenuates blood–brain barrier disruption after middle cerebral
artery occlusion in rats. Exp Neurol. 2012;237:453–63.
75. Bozimowski G. A review of nonsteroidal anti-inflammatory drugs. AANA J.
2015;83:425–33.
76. Patrono C. Cardiovascular effects of nonsteroidal anti-inflammatory drugs.
Curr Cardiol Rep. 2016;18:25.
77. Beltrán-Villalobos KL, Déciga-Campos M, Aguilar-Mariscal H, González-
Trujano ME, Martínez-Salazar MF, Ramírez-Cisneros MLÁ, et al.
Synergistic antinociceptive interaction of Syzygium aromaticum or
Rosmarinus officinalis coadministered with ketorolac in rats. Biomed
Pharmacother. 2017;94:858–64.
78. Hosseinzadeh H, Nourbakhsh M. Effect of Rosmarinus offcinalis L. aerial parts extract
on morphine withdrawal syndrome in mice. Phytother Res. 2003;17:938–41.
79. Martínez AL, González-Trujano ME, Chávez M, Pellicer F. Antinociceptive
effectiveness of triterpenes from rosemary in visceral nociception. J
Ethnopharmacol. 2012;142:28–34.
80. Liu BB, Luo L, Liu XL, Geng D, Li CF, Chen SM, et al. Essential oil of Syzygium
aromaticum reverses the deficits of stress-induced behaviors and hippocampal
p-ERK/p-CREB/brain-derived neurotrophic factor expression. Planta Med. 2015;
81:185–92.
81. Lee SH, Moon JY, Jung SJ, Kang JG, Choi SP, Jang JH. Eugenol inhibits the
GABAA current in trigeminal ganglion neurons. PLoS One. 2015;10:e0117316.
82. Abdelhalim A, Karim N, Chebib M, Aburjai T, Khan I, Johnston GA, et al.
Antidepressant, anxiolytic and antinociceptive activities of constituents from
Rosmarinus Officinalis. J Pharm Sci. 2015;18:448–59.
83. Martínez AL, González-Trujano ME, Chávez M, Pellicer F, Moreno J, López-
Muñoz FJ. Hesperidin produces antinociceptive response and synergistic
interaction with ketorolac in an arthritic gout-type pain in rats. Pharmacol
Biochem Behav. 2011;97:683–9.
84. Lima DF, Brandão MS, Moura JB, Leitão JM, Carvalho FA, Miúra LM.
Antinociceptive activity of the monoterpene –phellandrene in rodents:
possible mechanisms of action. J Pharm Pharmacol. 2012;64:283–92.
85. Verano J, González-Trujano ME, Déciga-Campos M, Ventura-Martínez R,
Pellicer F. Ursolic acid from Agastache mexicana aerial parts produces
antinociceptive activity involving TRPV1 receptors, cGMP and a serotonergic
synergism. Pharmacol Biochem Behav. 2013;110:255–64.
86. Poeckel D, Greiner C, Verhoff M, Rau O, Tausch L, Hörnig C. Carnosic acid
and carnosol potently inhibit human 5-lipoxygenase and suppress pro-
inflammatory responses of stimulated human polymorphonuclear
leukocytes. Biochem Pharmacol. 2008;76:91–7.
87. Harriott MM, Noverr MC. Candida albicans and Staphylococcus aureus form
polymicrobial biofilms: effects on antimicrobial resistance. Antimicrob
Agents Chemother. 2009;53:3914–22.
88. Ammons MC, Tripet BP, Carlson RP, Kirker KR, Gross MA, Stanisich JJ, et al.
Quantitative NMR metabolite profiling of methicillin-resistant and
methicillin-susceptible Staphylococcus aureus discriminates between biofilm
and planktonic phenotypes. J Proteome Res. 2014;13:2973–85.
89. Garrett TR, Bhakoo M, Zhang Z. Bacterial adhesion and biofilms on surfaces.
Prog Nat Sci. 2008;18:1049–56.
90. Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother. 2001;
45:999–1007.
91. de Oliveira JR, de Jesus D, Figueira LW, de Oliveira FE, Pacheco Soares C,
Camargo SE, Jorge AO, de Oliveira LD. Biological activities of Rosmarinus
officinalis L. (rosemary) extract as analyzed in microorganisms and cells. Exp
Biol Med (Maywood). 2017;242:625–34.
92. Silva DC, Lourenço AG, Ribeiro AE, Machado AA, Komesu MC, Motta AC.
Oral health management of 97 patients living with HIV/AIDS in Ribeirão
Preto, São Paulo. Brazil Braz Oral Res. 2015;29:1–6.
93. Zuanazzi D, Souto R, Mattos MB, Zuanazzi MR, Tura BR, Sansone C, et al.
Prevalence of potential bacterial respiratory pathogens in the oral cavity of
hospitalised individuals. Arch Oral Biol. 2010;55:21–8.
94. Zhang C, Du J, Peng Z. Correlation between Enterococcus faecalis and
persistent intraradicular infection compared with primary intraradicular
infection: a systematic review. J Endod. 2015;41:1207–13.
95. Gregoire S, Xiao J, Silva BB, Gonzalez I, Agidi PS, Klein MI, et al. Role of
glucosyltransferase B in interactions of Candida albicans with Streptococcus
mutans and with an experimental pellicle on hydroxyapatite surfaces. Appl
Environ Microbiol. 2011;77:6357–67.
96. Raghavendran K, Mylotte JM, Scannapieco FA. Nursing home-associated
pneumonia, hospital-acquired pneumonia and ventilator-associated
pneumonia: the contribution of dental biofilms and periodontal
inflammation. Periodontol 2000. 2007;44:164–77.
97. Klotz SA, Chasin BS, Powell B, Gaur NK, Lipke PN. Polymicrobial bloodstream
infections involving Candida species: analysis of patients and review of the
literature. Diagn Microbiol Infect Dis. 2007;59:401–6.
98. Cruz MR, Graham CE, Gagliano BC, Lorenz MC, Garsin DA. Enterococcus
faecalis inhibits hyphal morphogenesis and virulence of Candida albicans.
Infect Immun. 2013;81:189–200.
99. Parisotto TM, Steiner-Oliveira C, Silva CM, Rodrigues LK, Nobre-dos-Santos
M. Early childhood caries and mutans streptococci: a systematic review. Oral
Health Prev Dent. 2010;8:59–70.
100. Morales DK, Jacobs NJ, Rajamani S, Krishnamurthy M, Cubillos-Ruiz JR,
Hogan DA. Antifungal mechanisms by which a novel Pseudomonas
aeruginosa phenazine toxin kills Candida albicans in biofilms. Mol Microbiol.
2010;78:1379–92.
101. Sandasi M, Leonard CM, Viljoen AM. The in vitro antibiofilm activity of
selected culinary herbs and medicinal plants against listeria
monocytogenes. Lett Appl Microbiol. 2010;50:30–5.
102. Bavington C, Page C. Stopping bacterial adhesion: a novel approach to
treating infections. Respiration. 2005;72:335–44.
103. Nostro A, Sudano Roccaro A, Bisignano G, Marino A, Cannatelli MA,
Pizzimenti FC, et al. Effects of oregano, carvacrol and thymol on
Staphylococcus aureus and Staphylococcus epidermidis biofilms. J Med
Microbiol. 2007;56:519–23.
104. Lambert RJ, Skandamis PN, Coote PJ, Nychas GJ. A study of the minimum
inhibitory concentration and mode of action of oregano essential oil,
thymol and carvacrol. J Appl Microbiol. 2001;91:453–62.
105. Trombetta D, Castelli F, Sarpietro MG, Venuti V, Cristani M, Daniele C, et al.
Mechanisms of antibacterial action of three monoterpenes. Antimicrob
Agents Chemother. 2005;49:2474–8.
106. da Silva BN, Nakassugi LP, Faggion Pinheiro Oliveira J, Kohiyama CY, Mossini
SA, Grespan R, et al. Antifungal activity and inhibition of fumonisin
production by Rosmarinus officinalis L. essential oil in Fusarium verticillioides
(Sacc.) Nirenberg. Food Chem. 2015;166:330–6.
107. Li S, Myung K, Guse D, Donkin B, Proctor RH, Grayburn WS, et al. FvVE1
regulates filamentous growth, the ratio of microconidia to macroconidia and
cell wall formation in fusarium verticillioides. Mol Microbiol. 2006;62:1418–32.
108. Carmo ES, de Oliveira LE, de Souza EL, de Sousa FB. Effect of Cinnamomum
zeylanicum Blume essential oil on the growth and morphogenesis of some
potentially pathogenic aspergillus species. Braz J Microbiol. 2008;39:91–7.
109. Bendaha H, Yu L, Touzani R, Souane R, Giaever G, Nislow C, et al. New azole
antifungal agents with novel modes of action: synthesis and biological
studies of new tridentate ligands based on pyrazole and triazole. Eur J Med
Chem. 2011;46:4117–24.
110. Gauch LM, Silveira-Gomes F, Esteves RA, Pedrosa SS, Gurgel ES, Arruda AC,
et al. Effects of Rosmarinus officinalis essential oil on germ tube formation
by Candida albicans isolated from denture wearers. Rev Soc Bras Med Trop.
2014;47:389–91.
111. Pinto E, Hrimpeng K, Lopes G, Gonçalves SVMJ, Cavaleiro C, Salgueiro L.
Antifungal activity of Ferulago capillaris essential oil against Candida,
Cryptococcus,Aspergillus and dermatophyte species. Eur J Clin Microbiol
Infect Dis. 2013;40:375–80.
112. Soliman MM, Baiomy AA, Yassin MH. Molecular and histopathological study
on the ameliorative effects of curcumin against lead acetate-induced
hepatotoxicity and nephrototoxicity in Wistar rats. Biol Trace Elem Res. 2015;
167:91–102.
113. Mudipalli A. Lead hepatotoxicity & potential health effects. Indian J Med
Res. 2007;126:518–27.
114. Markowitz M. Lead poisoning. Pediatr Rev. 2000;21:327–35.
115. Iavicoli I, Carelli G, Stanek EJ, Castellino N, Calabrese EJ. Effects of low doses
of dietary lead on red blood cell production in male and female mice.
Toxicol Lett. 2003;137:193–9.
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 19 of 22

116. Shah SL, Altindag A. Alterations in the immunological parameters of Tench (Tinca
tinca L. 1758) after acute and chronic exposure to lethal and sublethal treatments
with mercury, cadmium and lead. Turk J Vet Anim Sci. 2005;29:1163–8.
117. Patra RC, Swarup D. Effect of antioxidant ascorbic acid, l-methionine or a
tocopherol alone or along with chelator on cardiac tissue of lead-treated
rats. Vet Arh. 2004;74:235–44.
118. Ademuyiwa O, Ugbaja RN, Rotimi SO, Abam E, Okediran BS, Dosumu OA, et
al. Erythrocyte acetylcholinesterase activity as a surrogate indicator of lead-
induced neurotoxicity in occupational lead exposure in Abeokuta. Nigeria
Environ Toxicol Pharmacol. 2007;24:183–8.
119. Teijón C, Olmo R, Blanco D, Romero A, Teijón JM. Low doses of lead: effects on
reproduction and development in rats. Biol Trace Elem Res. 2006;111:151–65.
120. Landrigan PJ, Boffetta P, Apostoli P. The reproductive toxicity and
carcinogenicity of lead: a critical review. Am J Ind Med. 2000;38:231–43.
121. Adikwu E, Deo O, Geoffrey O-BP, Enimeya DA. Lead organ and tissue toxicity:
roles of mitigating agents (part 1). Br J Med Med Res. 2013;4:232–40.
122. Porru S, Alessio L. The use of chelating agents in occupational lead
poisoning. Occup Med (Lond). 1996;46:41–8.
123. Mohamed WA, Abd-Elhakim YM, Farouk SM. Protective effects of ethanolic
extract of rosemary against lead-induced hepato-renal damage in rabbits.
Exp Toxicol Pathol. 2016;68:451–61.
124. Jurczuk M, Brzóska M, Moniuszko-Jakoniuk J. Hepatic and renal
concentrations of vitamins E and C in lead and ethanol-exposed rats: an
assessment of their involvement in the mechanisms of peroxidative
damage. Food Chem Toxicol. 2007;45:1478–86.
125. Abd El Rahiem A, Yassin MM, Nahed M, Rokaya M. Blood, serum glucose
and renal parameters in lead-loaded albino rats and treatment with some
chelating agents and natural oils. Turk J Biol. 2007;31:25–34.
126. Chmielnicka J, Zareba G, Nasiadek M. Combined effect of tin and lead on
heme biosynthesis in rats. Ecotoxicol Environ Safe. 1994;29:165–73.
127. Maurer J, Rebbapragada V, Borson S, Goldstein R, Kunik ME, Yohannes AM,
et al. Anxiety and depression in COPD: current understanding, unanswered
questions, and research needs. Chest. 2008;134:43S–56S.
128. Mann JJ. The medical management of depression. N Engl J Med. 2005;353:
1819–34 76.
129. Schulz P, Macher JP. The clinical pharmacology of depressive states.
Dialogues Clin Neurosci. 2002;4:47–56.
130. Koenig AM, Thase ME. First-line pharmacotherapies for depression - what is
the best choice? Pol Arch Med Wewn. 2009;119:478–86.
131. Akhondzadeh S, Kashani L, Fotouhi A, Jarvandi S, Mobaseri M, Moin M, et al.
Comparison of Lavandula angustifolia mill. Tincture and imipramine in the
treatment of mild to moderate depression: a double-blind, randomized trial.
Prog Neuro-Psychopharmacol Biol Psychiatry. 2003;27:123–7.
132. Abadi MNA, Mortazavi M, Kalani N, Marzouni HZ, Kooti W, Ali-Akbari S.
Eeffect of hydroalcoholic extract of Rosmarinus officinalis L. leaf on anxiety
in mice. J Evid Based Complementary Altern Med. 2016;21:NP85–90.
133. Rezaie A, Mosavi G, Ahmadizadeh C, Jafari B. Study of sedative,
preanaesthetic and anti-anxiety effects of Rosa damascene herbal extract in
comparison with diazepam in rat. Tehran Univ Med J. 2011;69:179–84.
134. Marder M, Paladini AC. GABA(a)-receptor ligands of flavonoid structure. Curr
Top Med Chem. 2002;2:853–67.
135. Niksokhan M, Hedarieh N, Najafifard M, Najafifard M. Effect of hydro-alcholic
extract of Pimpinella anisum seed on anxiety in male rat. J Gorgan Univ
Med Sci. 2014;16:28–33.
136. Kumar A, Agarwal P, Shakya A, Thakur AK, Kumar V. Key role of carnosic acid
in the anxiolytic-like activity of Rosmarinus officinalis Linn. J Pharmacol Clin
Toxicol. 2013;1:1013.
137. Vaupel P. Tumor microenvironmental physiology and its implications for
radiation oncology. Semin Radiat Oncol. 2004;14:198–206.
138. Heldin CH, Rubin K, Pietras K, Ostman A. High interstitial fluid pressure—an
obstacle in cancer therapy. Nat Rev Cancer. 2004;4:806–13.
139. Aukland K, Reed RK. Interstitial-lymphatic mechanisms in the control of
extracellular fluid volume. Physiol Rev. 1993;73:1–78.
140. Jain RK. Physiological barriers to delivery of monoclonal antibodies and
other macromolecules in tumors. Cancer Res. 1990;50:814–9s.
141. Cardenas-Navia LI, Yu D, Braun RD, Brizel DM, Secomb TW, Dewhirst MW.
Tumor-dependent kinetics of partial pressure of oxygen fluctuations during
air and oxygen breathing. Cancer Res. 2004;64:6010–7.
142. van den Berg AP, Wike-Hooley JL, van den Berg-Blok AE, van der Zee J,
Reinhold HS. Tumour pH in human mammary carcinoma. Eur J Cancer Clin
Oncol 1982;18:457–462.
143. Yamagata M, Hasuda K, Stamato T, Tannock IF. The contribution of lactic
acid to acidification of tumours: studies of variant cells lacking lactate
dehydrogenase. Br J Cancer. 1998;77:1726–31.
144. Helmlinger G, Sckell A, Dellian M, Forbes NS, Jain RK. Acid production in
glycolysis-impaired tumors provides new insights into tumor metabolism.
Clin Cancer Res. 2002;8:1284–91.
145. Curti BD, Urba WJ, Alvord WG, Janik JE, Smith JW 2nd, Madara K, et al.
Interstitial pressure of subcutaneous nodules in melanoma and lymphoma
patients: changes during treatment. Cancer Res. 1993;53:2204–7.
146. Mohindra JK, Rauth AM. Increased cell killing by metronidazole and
nitrofurazone of hypoxic compared to aerobic mammalian cells. Cancer
Res. 1976;36:930–6.
147. Koch S, Mayer F, Honecker F, Schittenhelm M, Bokemeyer C. Efficacy of
cytotoxic agents used in the treatment of testicular germ cell tumours under
normoxic and hypoxic conditions in vitro. Br J Cancer. 2003;89:2133–9.
148. Green SL, Freiberg RA, Giaccia AJ. p21(Cip1) and p27(Kip1) regulate cell
cycle reentry after hypoxic stress but are not necessary for hypoxia-induced
arrest. Mol Cell Biol. 2001;21:1196–206.
149. Gerweck LE, Kozin SV, Stocks SJ. The pH partition theory predicts the
accumulation and toxicity of doxorubicin in normal and low-pH-adapted
cells. Br J Cancer. 1999;79:838–42.
150. Mahoney BP, Raghunand N, Baggett B, Gillies RJ. Tumor acidity, ion trapping
and chemotherapeutics. I. Acid pH affects the distribution of chemotherapeutic
agents in vitro. Biochem Pharmacol. 2003;66:1207–18.
151. Hurwitz SJ, Terashima M, Mizunuma N, Slapak CA. Vesicular anthracycline
accumulation in doxorubicin-selected U-937 cells: participation of lysosomes.
Blood. 1997;89:3745–54.
152. Amar Y, Meddah B, Bonacorsi I, Costa G, Pezzino G, Saija A, et al. Phytochemicals,
antioxidant and antiproliferative properties of Rosmarinus officinalis LonU937
and CaCo-2 cells. Iran J Pharm Res. 2017;16:315–27.
153. Karimi N, Rashedi J, Mahdavi Poor B, Arabi S, Ghorbani M, Tahmasebpour N,
et al. Cytotoxic effect of rosemary extract on gastric adenocarcinoma (AGS)
and esophageal squamous cell carcinoma (KYSE30) cell lines. Gastroenterol
Hepatol Bed Bench. 2017;10:102–7.
154. Moore J, Megaly M, MacNeil AJ, Klentrou P, Tsiani E. Rosemary extract
reduces Akt/mTOR/p70S6K activation and inhibits proliferation and survival
of A549 human lung cancer cells. Biomed Pharmacother. 2016;83:725–32.
155. Zakaria ZA, Rofiee MS, Mohamed AM, Teh LK, Salleh MZ. In-vitro
antiproliferative and antioxidant activities and total phenolic contents of the
extracts of Melastoma malabathricum leaves. J Acupunct Meridian Stud.
2011;4:248–56.
156. Manosroi J, Boonpisuttinant K, Manosroi W, Manosroi A. Anti-proliferative
activities on HeLa cancer cell line of Thai medicinal plant recipes selected
from MANOSROI II database. J Ethnopharmacol. 2012;142:422–31.
157. Nagendra-Prasad K, Hao J, Shi J, Liu T, Li J, Wei X, et al. Antioxidant and
anticancer activities of high pressure-assisted extract of longan (Dimocarpus
longan Lour.) fruit pericarp. Innov Food Sci Emer Tech. 2009;10:413–9.
158. Olsson ME, Andersson CS, Oredsson S, Berglund RH, Gustavsson KE.
Antioxidant levels and inhibition of cancer cell proliferation in vitro by
extracts from organically and conventionally cultivated strawberries. J Agric
Food Chem. 2006;54:1248–55.
159. Olsson ME, Gustavsson KE, Andersson S, Nilsson A, Duan RD. Inhibition of
cancer cell proliferation in vitro by fruit and berry extracts and correlations
with antioxidant levels. J Agric Food Chem. 2004;52:7264–71.
160. Sharif T, Alhosin M, Auger C, Minker C, Kim JH, Etienne-Selloum N, et al.
Aronia melanocarpa juice induces a redox-sensitive p73-related caspase 3-
dependent apoptosis in human leukemia cells. PLoS One. 2012;7:e32526.
161. Juan ME, Wenzel U, Daniel H, Planas JM. Resveratrol induces apoptosis
through ROS-dependent mitochondria pathway in HT-29 human colorectal
carcinoma cells. J Agric Food Chem. 2008;56:4813–8.
162. Cheng AC, Lee MF, Tsai ML, Lai CS, Lee JH, Ho CT, et al. Rosmanol potently
induces apoptosis through both the mitochondrial apoptotic pathway and
death receptor pathway in human colon adenocarcinoma COLO 205 cells.
Food Chem Toxicol. 2011;49:485–93.
163. Lo AH, Liang YC, Lin-Shiau SY, Ho CT, Lin JK. Carnosol, an antioxidant in
rosemary, suppresses inducible nitric oxide synthase through down-
regulating nuclear factor-kappaB in mouse macrophages. Carcinogenesis.
2002;23:983–91.
164. Kim MY, Mauro S, Gévry N, Lis JT, Kraus WL. NAD+−dependent modulation
of chromatin structure and transcription by nucleosome binding properties
of PARP-1. Cell. 2004;119:803–14.
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 20 of 22

165. Nosseri C, Coppola S, Ghibelli L. Possible involvement of poly (ADP-ribosyl)
polymerase in triggering stress-induced apoptosis. Exp Cell Res. 1994;121:367–73.
166. Brognard J, Clark AS, Ni Y, Dennis PA. Akt/protein kinase B is constitutively
active in non-small cell lung cancer cells and promotes cellular survival and
resistance to chemotherapy and radiation. Cancer Res. 2001;61:3986–97.
167. Valdés A, García-Cañas V, Rocamora-Reverte L, Gómez-Martínez A, Ferragut
JA, Cifuentes A. Effect of rosemary polyphenols on human colon cancer
cells: transcriptomic profiling and functional enrichment analysis. Genes
Nutr. 2013;8:43–60.
168. Kim G, Dasagrandhi C, Kang EH, Eom SH, Kim YM. In vitro antibacterial and
early stage biofilm inhibitory potential of an edible chitosan and its
phenolic conjugates against Pseudomonas aeruginosa and Listeria
monocytogenes. 3 Biotech. 2018;8:439.
169. Liu Q, Tang GY, Zhao CN, Feng XL, Xu XY, Cao SY, et al. Comparison of
antioxidant activities of different grape varieties. Molecules. 2018;23(10):1–17.
170. Kabala-Dzik A, Rzepecka-Stojko A, Kubina R, Wojtyczka RD, Buszman E, Stojko J.
Caffeic acid versus caffeic acid phenethyl ester in the treatment of breast cancer
MCF-7 cells: migration rate inhibition. Integr Cancer Ther. 2018;17:1247–59.
171. von Schönfeld C, Huber R, Trittler R, Kammerer B, Garcia-Käufer M,
Gründemann C. Rosemary has immunosuppressant activity mediated
through the STAT3 pathway. Complement Ther Med. 2018;40:165–70.
172. Mu HN, Li Q, Fan JY, Pan CS, Liu YY, Yan L, et al. Caffeic acid attenuates rat
liver injury after transplantation involving PDIA3-dependent regulation of
NADPH oxidase. Free Radic Biol Med. 2018;129:202–14.
173. Min J, Shen H, Xi W, Wang Q, Yin L, Zhang Y, et al. Synergistic anticancer
activity of combined use of caffeic acid with paclitaxel enhances apoptosis
of non-small-cell lung cancer H1299 cells in vivo and in vitro. Cell Physiol
Biochem. 2018;48:1433–42.
174. Liu D, Wang B, Zhu Y, Yan F, Dong W. Carnosic acid regulates cell
proliferation and invasion in chronic myeloid leukemia cancer cells via
suppressing microRNA-708. J Buon. 2018;23:741–6.
175. Albalawi A, Alhasani RHA, Biswas L, Reilly J, Akhtar S, Shu X. Carnosic acid
attenuates acrylamide-induced retinal toxicity in zebrafish embryos. Exp Eye
Res. 2018;175:103–14.
176. Lin KI, Lin CC, Kuo SM, Lai JC, Wang YQ, You HL, et al. Carnosic acid
impedes cell growth and enhances anticancer effects of carmustine and
lomustine in melanoma. Biosci Rep. 2018;38(4):1–11.
177. Wang LC, Wei WH, Zhang XW, Liu D, Zeng KW, Tu PF. An integrated
proteomics and bioinformatics approach reveals the anti-inflammatory
mechanism of carnosic acid. Front Pharmacol. 2018;9:370.
178. Ercan P, El SN. Bioaccessibility and inhibitory effects on digestive enzymes of
carnosic acid in sage and rosemary. Int J Biol Macromol. 2018;115:933–9.
179. Song HM, Li X, Liu YY, Lu WP, Cui ZH, Zhou L, et al. Carnosic acid protects
mice from high-fat diet-induced NAFLD by regulating MARCKS. Int J Mol
Med. 2018;42:193–207.
180. de Lima ME, Ceolin Colpo AZ, Maya-López M, Rangel-López E, Becerril-
Chávez H, Galván-Arzate S, et al. Comparing the effects of chlorogenic acid
and Ilex paraguariensis extracts on different markers of brain alterations in
rats subjected to chronic restraint stress. Neurotox Res. 2018. https://doi.org/
10.1007/s12640-018-9963-6.
181. Zhang T, Chen S, Chen L, Zhang L, Meng F, Sha S, et al. Chlorogenic acid
ameliorates lead-induced renal damage in mice. Biol Trace Elem Res. 2018.
https://doi.org/10.1007/s12011-018-1508-6.
182. Vukelic I, Detel D, Pucar LB, Potocnjak I, Buljevic S, Domitrovic R.
Chlorogenic acid ameliorates experimental colitis in mice by suppressing
signaling pathways involved in inflammatory response and apoptosis. Food
Chem Toxicol. 2018;121:140–50.
183. Gong XX, Su XS, Zhan K, Zhao GQ. The protective effect of chlorogenic acid
on bovine mammary epithelial cells and neutrophil function. J Dairy Sci.
2018;101:10089–97.
184. Khwaza V, Oyedeji OO, Aderibigbe BA. Antiviral activities of oleanolic acid and its
analogues. Molecules. 2018;23(9). https://doi.org/10.3390/molecules23092300.
185. Zhang W, Feng J, Cheng B, Lu Q, Chen X. Oleanolic acid protects against
oxidative stress-induced human umbilical vein endothelial cell injury by
activating AKT/eNOS signaling. Mol Med Rep. 2018;18:3641–8.
186. Ali MS, Ahmed G, Mesaik MA, Shah MR, Lateef M, Ali MI. Facile one-pot
syntheses of new C-28 esters of oleanolic acid and studies on their
antiproliferative effect on T cells. Z Naturforsch C. 2018;73:417–21.
187. Kim GJ, Jo HJ, Lee KJ, Choi JW, An JH. Oleanolic acid induces p53-
dependent apoptosis via the ERK/JNK/AKT pathway in cancer cell lines in
prostatic cancer xenografts in mice. Oncotarget. 2018;9:26370–86.
188. Bai X, Lai T, Zhou T, Li Y, Li X, Zhang H. In vitro antioxidant activities of phenols
and oleanolic acid from mango peel and their cytotoxic effect on a549 cell
line. Molecules. 2018;23(6). https://doi.org/10.3390/molecules23061395.
189. Cui HY, Zhang XJ, Yang Y, Zhang C, Zhu CH, Miao JY, et al. Rosmarinic acid
elicits neuroprotection in ischemic stroke via Nrf2 and heme oxygenase 1
signaling. Neural Regen Res. 2018;13:2119–28.
190. Yao Y, Mao J, Xu S, Zhao L, Long L, Chen L, et al. Rosmarinic acid inhibits
nicotine-induced C-reactive protein generation by inhibiting NLRP3
inflammasome activation in smooth muscle cells. J Cell Physiol. 2018.
https://doi.org/10.1002/jcp.27046.
191. Radziejewska I, Supruniuk K, Nazaruk J, Karna E, Poplawska B, Bielawska A, et al.
Rosmarinic acid influences collagen, MMPs, TIMPs, glycosylation and MUC1 in
CRL-1739 gastric cancer cell line. Biomed Pharmacother. 2018;107:397–407.
192. Makhathini KB, Mabandla MV, Daniels WMU. Rosmarinic acid reverses the
deleterious effects of repetitive stress and tat protein. Behav Brain Res. 2018;
353:203–9.
193. Ma ZJ, Yan H, Wang YJ, Yang Y, Li XB, Shi AC, et al. Proteomics analysis
demonstrating rosmarinic acid suppresses cell growth by blocking the glycolytic
pathway in human HepG2 cells. Biomed Pharmacother. 2018;105:334–49.
194. Tsukamoto Y, Ikeda S, Uwai K, Taguchi R, Chayama K, Sakaguchi T, et
al. Rosmarinic acid is a novel inhibitor for hepatitisBvirusreplication
targeting viral epsilon RNA-polymerase interaction. PLoS One. 2018;
13(5):e0197664.
195. Kahnt M, Fischer Née Heller L, Al-Harrasi A, Csuk R. Ethylenediamine derived
carboxamides of betulinic and ursolic acid as potential cytotoxic agents.
Molecules. 2018;23(10). https://doi.org/10.3390/molecules23102558.
196. Yadav D, Mishra BN, Khan F. 3D-QSAR and docking studies on ursolic acid
derivatives for anticancer activity based on bladder cell line T24 targeting
NF-kB pathway inhibition. J Biomol Struct Dyn. 2018:1–33. https://doi.org/10.
1080/07391102.2018.1528888.
197. Cheng M, Liang XH, Wang QW, Deng YT, Zhao ZX, Liu XY. Ursolic acid
prevents retinoic acid-induced bone loss in rats. Chin J Integr Med. 2018.
https://doi.org/10.1007/s11655-018-3050-y.
198. Abu-Gharbieh E, Shehab NG, Almasri IM, Bustanji Y. Antihyperuricemic and
xanthine oxidase inhibitory activities of Tribulus arabicus and its isolated
compound, ursolic acid: in vitro and in vivo investigation and docking
simulations. PLoS One. 2018;13:e0202572.
199. Lee KC, Chen YL, Lin PY, Chuang WL. Ursolic acid-induced apoptosis via
regulation of the PI3K/Akt and MAPK signaling pathways in Huh-7 cells.
Molecules. 2018;23(8). https://doi.org/10.3390/molecules23082016.
200. Mourya A, Akhtar A, Ahuja S, Sah SP, Kumar A. Synergistic action of ursolic
acid and metformin in experimental model of insulin resistance and related
behavioral alterations. Eur J Pharmacol. 2018;835:31–40.
201. Xu HL, Wang XT, Cheng Y, Zhao JG, Zhou YJ, Yang JJ, et al. Ursolic acid
improves diabetic nephropathy via suppression of oxidative stress and
inflammation in streptozotocin-induced rats. Biomed Pharmacother. 2018;
105:915–21.
202. Nguyen HN, Ahn YJ, Medina EA, Asmis R. Dietary 23-hydroxy ursolic acid
protects against atherosclerosis and obesity by preventing dyslipidemia-
induced monocyte priming and dysfunction. Atherosclerosis. 2018;275:333–41.
203. de Sousa Eduardo L, Farias TC, Ferreira SB, Ferreira PB, Lima ZN, Ferreira SB.
Antibacterial activity and time-kill kinetics of positive enantiomer of a-
pinene against strains of Staphylococcus aureus and Escherichia coli. Curr
Top Med Chem. 2018;18:917–24.
204. Ložiene K, Švediene J, Paškevicius A, Raudoniene V, Sytar O, Kosyan A.
Influence of plant origin natural a-pinene with different enantiomeric
composition on bacteria, yeasts and fungi. Fitoterapia. 2018;127:20–4.
205. Bouzenna H, Hfaiedh N, Giroux-Metges MA, Elfeki A, Talarmin H. Potential
protective effects of alpha-pinene against cytotoxicity caused by aspirin in
the IEC-6 cells. Biomed Pharmacother. 2017;93:961–8.
206. Memariani Z, Sharifzadeh M, Bozorgi M, Hajimahmoodi M, Farzaei MH,
Gholami M, et al. Protective effect of essential oil of Pistacia atlantica Desf.
On peptic ulcer: role of a-pinene. J Tradit Chin Med. 2017;37:57–63.
207. Lin YH, Kuo JT, Chen YY, Kumar KJS, Lo CP, Lin CC, et al.
Immunomodulatory effects of the stout camphor medicinal mushroom,
Taiwanofungus camphoratus (Agaricomycetes)-based health food product in
mice. Int J Med Mushrooms. 2018;20:849–58.
208. Carvalho MFNN, Botelho do Rego AM, Galvão AM, Herrmann R, Marques F.
Search for cytotoxic compounds against ovarian cancer cells: synthesis,
characterization and assessment of the activity of new camphor carboxylate
and camphor carboxamide silver complexes. J Inorg Biochem. 2018;188:88–95.
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 21 of 22

209. Kuranov SO, Tsypysheva IP, Khvostov MV, Zainullina LF, Borisevich SS,
Vakhitova YV, et al. Synthesis and evaluation of camphor and cytisine-based
cyanopyrrolidines as DPP-IV inhibitors for the treatment of type 2 diabetes
mellitus. Bioorg Med Chem. 2018;26:4402–9.
210. Leitão JH, Sousa SA, Leite SA, Carvalho MFNN. Silver camphor imine
complexes: novel antibacterial compounds from old medicines. Antibiotics
(Basel). 2018;7(3). https://doi.org/10.3390/antibiotics7030065.
211. Manoharan RK, Lee JH, Lee J. Antibiofilm and antihyphal activities of cedar
leaf essential oil, camphor, and fenchone derivatives against Candida
albicans. Front Microbiol. 2017;8:1476.
212. Aliebrahimi S, Kouhsari SM, Arab SS, Shadboorestan A, Ostad SN.
Phytochemicals, withaferin a and carnosol, overcome pancreatic cancer
stem cells as c-met inhibitors. Biomed Pharmacother. 2018;106:1527–36.
213. Zheng Y, Zhang Y, Zheng Y, Zhang N. Carnosol protects against renal
ischemia-reperfusion injury in rats. Exp Anim. 2018;67:545–53.
214. Ramírez J, Gilardoni G, Ramón E, Tosi S, Picco AM, Bicchi C, et al.
Phytochemical study of the ecuadorian species Lepechinia mutica (Benth.)
epling and high antifungal activity of carnosol against pyricularia oryzae.
Pharmaceuticals (Basel). 2018;11(2). https://doi.org/10.3390/ph11020033.
215. Lo YC, Lin YC, Huang YF, Hsieh CP, Wu CC, Chang IL, et al. Carnosol-
induced ROS inhibits cell viability of human osteosarcoma by apoptosis and
autophagy. Am J Chin Med. 2017;45:1761–72.
216. Oliviero F, Scanu A, Zamudio-Cuevas Y, Punzi L, Spinella P. Anti-inflammatory
effects of polyphenols in arthritis. J Sci Food Agric. 2018;98:1653–9.
217. Lee DY, Hwang CJ, Choi JY, Park MH, Song MJ, Oh KW, et al. Inhibitory
effect of carnosol on phthalic anhydride-induced atopic dermatitis via
inhibition of stat3. Biomol Ther (Seoul). 2017;25(5):535–44.
218. Samarghandian S, Borji A, Farkhondeh T. Evaluation of antidiabetic activity
of carnosol (phenolic diterpene in rosemary) in streptozotocin-induced
diabetic rats. Cardiovasc Hematol Disord Drug Targets. 2017;17:11–7.
219. Sampath S, Subramani S, Janardhanam S, Subramani P, Yuvaraj A, Chellan R.
Bioactive compound 1,8-cineole selectively induces G2/M arrest in A431
cells through the upregulation of the p53 signaling pathway and molecular
docking studies. Phytomedicine. 2018;46:57–68.
220. Merghni A, Noumi E, Hadded O, Dridi N, Panwar H, Ceylan O, et al.
Assessment of the antibiofilm and antiquorum sensing activities of
Eucalyptus globulus essential oil and its main component 1,8-cineole
against methicillin-resistant Staphylococcus aureus strains. Microb
Pathog. 2018;118:74–80.
221. Yadav N, Chandra H. Suppression of inflammatory and infection responses
in lung macrophages by eucalyptus oil and its constituent 1,8-cineole: role
of pattern recognition receptors TREM-1 and NLRP3, the MAP kinase regulator
MKP-1, and NF?B. PLoS One. 2017;12(11):e0188232.
222. Martins AOBPB, Rodrigues LB, Cesário FRAS, de Oliveira MRC, Tintino CDM,
Castro FFE, et al. Anti-edematogenic and anti-inflammatory activity of the
essential oil from Croton rhamnifolioides leaves and its major constituent
1,8-cineole (eucalyptol). Biomed Pharmacother. 2017;96:384–95.
223. Melo Júnior JM, Damasceno MB, Santos SA, Barbosa TM, Araújo JR, Vieira-
Neto AE, et al. Acute and neuropathic orofacial antinociceptive effect of
eucalyptol. Inflammopharmacology. 2017;25:247–54.
224. Lai YN, Li Y, Fu LC, Zhao F, Liu N, Zhang FX, et al. Combinations of 1,8-
cineol and oseltamivir for the treatment of influenza virus a (H3N2)
infection in mice. J Med Virol. 2017;89:1158–67.
225. Petiwala SM, Johnson JJ. Diterpenes from rosemary (Rosmarinus officinalis):
defining their potential for anti-cancer activity. Cancer Lett. 2015;367:93–102.
226. Novato T, Gomes GA, Zeringóta V, Franco CT, de Oliveira DR, Melo D, et al.
In vitro assessment of the acaricidal activity of carvacrol, thymol, eugenol
and their acetylated derivatives on Rhipicephalus microplus (Acari: Ixodidae).
Vet Parasitol. 2018;260:1–4.
227. Leal Pinto SM, Herrera Sandoval LV, Vargas LY. In vitro susceptibility of
Microsporum spp. and mammalian cells to Eugenia caryophyllus essential oil,
eugenol and semisynthetic derivatives. Mycoses. 2018. https://doi.org/10.
1111/myc.12844.
228. Pal D, Sur S, Roy R, Mandal S, Kumar Panda C. Epigallocatechin gallate in
combination with eugenol or amarogentin shows synergistic chemotherapeutic
potential in cervical cancer cell line. J Cell Physiol. 2018;234:825–36.
229. Das A, Harshadha K, Dhinesh Kannan SK, Hari Raj K, Jayaprakash B. Evaluation
of therapeutic potential of eugenol-a natural derivative of Syzygium
aromaticum on cervical cancer. Asian Pac J Cancer Prev. 2018;19(7):1977–85.
230. Magalhães CB, Casquilho NV, Machado MN, Riva DR, Travassos LH, Leal-
Cardoso JH, et al. The anti-inflammatory and anti-oxidative actions of
eugenol improve lipopolysaccharide-induced lung injury. Respir Physiol
Neurobiol. 2019;259:30–6.
231. Kong X, Huo G, Liu S, Li F, Chen W, Jiang D. Luteolin suppresses
inflammation through inhibiting cAMP-phosphodiesterases activity and
expression of adhesion molecules in microvascular endothelial cells.
Inflammopharmacology. 2018. https://doi.org/10.1007/s10787-018-0537-2.
232. Jo BG, Park NJ, Jegal J, Choi S, Lee SW, Yi LW, et al. Stellera chamaejasme
and its main compound luteolin 7-o-glucoside alleviates skin lesions in
oxazolone- and 2,4-dinitrochlorobenzene-stimulated murine models of
atopic dermatitis. Planta Med. 2018. https://doi.org/10.1055/a-0746-8698.
233. Liao Y, Xu Y, Cao M, Huan Y, Zhu L, Jiang Y, et al. Luteolin induces
apoptosis and autophagy in mouse macrophage ANA-1 cells via the Bcl-2
pathway. J Immunol Res. 2018;2018:4623919.
234. Tagousop CN, Tamokou JD, Ekom SE, Ngnokam D, Voutquenne-Nazabadioko
L. Antimicrobial activities of flavonoid glycosides from Graptophyllum
grandulosum and their mechanism of antibacterial action. BMC
Complement Altern Med. 2018;18(1):252.
235. Dong X, Zhang J, Yang F, Wu J, Cai R, Wang T, et al. Effect of luteolin on
the methylation status of the OPCML gene and cell growth in breast cancer
cells. Exp Ther Med. 2018;16(4):3186–94.
236. Elmazoglu Z, Yar Saglam AS, Sonmez C, Karasu C. Luteolin protects
microglia against rotenone-induced toxicity in a hormetic manner through
targeting oxidative stress response, genes associated with Parkinson's
disease and inflammatory pathways. Drug Chem Toxicol. 2018:1–8. https://
doi.org/10.1080/01480545.2018.1504961.
237. Jing Z, Wang C, Yang Q, Wei X, Jin Y, Meng Q, et al. Luteolin attenuates
glucocorticoid-induced osteoporosis by regulating ERK/Lrp-5/GSK-3ß
signaling pathway in vivo and in vitro. J Cell Physiol. 2018. https://doi.org/
10.1002/jcp.27252.
de Oliveira et al. Journal of Biomedical Science (2019) 26:5 Page 22 of 22