Biomedicine & Pharmacotherapy 84 (2016) 569–582

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Relevance of carnosic acid to the treatment of several health disorders:

Molecular targets and mechanisms
Sana Bahria,b,* , Saloua Jameleddinea , Vadim Shlyonskyb
a
Laboratory of Physiology, Faculty of Medicine of Tunis, University of Tunis El Manar, La Rabta 1007, Tunis, Tunisia
b
Laboratory of Physiopathology and Pharmacology, Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium

A R T I C L E I N F O A B S T R A C T

Article history:
Received 26 June 2016 Carnosic acid is a phenolic diterperne compound found in abundance in sage and rosemary, which are
Received in revised form 29 August 2016 both widely used in traditional medicine. Research over the past decade indicates that carnosic acid has
Accepted 18 September 2016 multiple bioactive properties including antioxidant, anti-inflammatory and anticancer activities among
others. This review summarizes the current in vitro and in vivo data about the efficacy of carnosic acid in
Keywords: the prevention or treatment of various experimental health disorders. The analysis of the literature
Carnosic acid allows an insight into the participation of numerous signaling pathways modulated by carnosic acid, into
Antioxidant its synergistic potential and, thus, into the divergence in cellular mechanisms of action of this molecule.
Anti-inflammatory
ã 2016 Elsevier Masson SAS. All rights reserved.
Antitumor

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
2. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
3. Bioavailability and toxicity of carnosic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
4. Anti-cancer effect of carnosic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
4.1. Carnosic acid and colorectal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
4.2. Carnosic acid and liver cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
4.3. Carnosic acid and renal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
4.4. Carnosic acid and brain cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
4.5. Carnosic acid and leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
4.6. Others anticancer properties of carnosic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573
5. Carnosic acid and experimental disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
5.1. Carnosic acid and liver injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
5.2. Carnosic acid and cardiac injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
5.3. Carnosic acid and atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
5.4. Carnosic acid and brain injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
5.4.1. Carnosic acid and Alzheimer disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
5.4.2. Carnosic acid and Parkinson disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
5.4.3. Carnosic acid and retinal degeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
5.4.4. Carnosic acid protects from other neuronal agressions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
5.5. Canosic acid and obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
5.6. Carnosic acid and gastric lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
5.7. Carnosic acid and skin lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
5.8. Other possible therapeutic applications of carnosic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
6. Carnosic acid synergizes with other natural compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
7. Rosmarinic acid and carnosic acid: possible connection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578
8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578

* Corresponding author at: Laboratory of Physiology, Faculty of Medicine of Tunis, University of Tunis El Manar, La Rabta 1007, Tunis, Tunisia.
E-mail address: bahrisana88@gmail.com (S. Bahri).

http://dx.doi.org/10.1016/j.biopha.2016.09.067
0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
570 S. Bahri et al. / Biomedicine & Pharmacotherapy 84 (2016) 569–582

Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579

Ethical responsibilities protection of people and animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
Confidentiality of data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579

1. Introduction that this molecule exerts a dual protective role in rosemary plant
against environmental constraints by capturing free radicals in
Currently, research on bioactive substances of plant or animal chloroplasts and by preserving the stability of cell membranes [4].
origin earned a growing interest in numerous industrial sectors, Detection of the anticancer activity of CA was among the first
including cosmetics, pharmaceutics and food processing. Notably, research work carried out. CA exerted an antiproliferative action in
plants are rich source of biomolecules generally well tolerated by leukemic cells without induction of apoptotic or necrotic cell death
the human body. Many of these biomolecules served as natural [5]. This study demonstrated that CA could cooperate synergisti-
remedies since ancient times. The enormous technical progress, cally with other natural anticancer compounds, such as vitamin D
discoveries and innovations, which promoted industrialization, and retinoic acid metabolites, to potentiate anticancer effects.
helped pharmaceutical companies to identify natural substances Later on, consequent studies of CA in the field of oncology took a
obtained from plants and to determine their therapeutic potential. considerable place both in vivo and in vitro. However, studies in
The term “medicinal plant” characterizes species that contain other health areas demonstrate that CA is clearly promiscuous
active compounds, which belong mainly to the category of molecule that is useful in the treatment of many other human
secondary metabolites (polyphenols, essential oils) known for diseases.
their medicinal properties. However, caution should be taken as to In this review, we will focus on the biological effects of
what part of the plant to use, since they can differ in characteristics vegetable extracts rich in CA or pure CA that were demonstrated by
and contain more than one active compound in different different research teams. We will assess efficacy of CA in various
proportions. Therefore, plant usage requires a real knowledge to physiological assays and clinical trials published till now.
avoid any risk of poisoning.
Carnosic acid (CA) was discovered first by Linde in Salvia 2. Methods
officinalis L. [1], then by Wenkert et al. [2] in Rosmarinus officinalis L
leaves. Despite the fact that sage and rosemary are hailed since In order to explore available information about CA beneficial
ancient times for their therapeutic properties, the exploration of effects for the treatment/prevention of various diseases, we have
the mechanisms of CA action began only in the early 2000s. Over collected data published between August 2001 and April 2016 in
the last decade, several research teams studied the pharmacologi- the Pubmed database using the following appropriate combina-
cal properties of CA, demonstrating that this molecule may have tions of MeSH (Medical Subject Heading) terms: carnosic acid,
clinical applications for various human diseases. Indeed, studies carnosic acid antioxidant, carnosic acid cancer, carnosic acid brain,
using rosemary extract showed that its properties are closely carnosic acid obesity, carnosic acid breast, carnosic acid liver,
related to its phenolic constituents, especially the most abundant rosemary carnosic acid.
compounds, carnosic acid and rosmarinic acid. On the other hand,
consumption of the whole plant leads to the ingestion of products 3. Bioavailability and toxicity of carnosic acid
other than the active ingredient sought. This requires the
extraction and purification of the active compound in order to At present, there are only two studies on bioavailability of
reveal its effects, to know the dose administered and to avoid the carnosic acid. First, it was reported that 6 h after oral administra-
risk of overdose that can lead to toxicity. tion in rats (64.3  5.8 mg/kg), the bioavailability of CA in its free
Carnosic acid (C20H28O4, Fig. 1), a phenolic diterpene that form was 40.1%, and its excretion in the feces after 24 h was
belongs to the terpene class of secondary metabolites [3], is 15.6  8.2% [6]. CA is absorbed into the bloodstream after oral
localized in rosemary leaves, more precisely in chloroplasts of administration and its traces were found in the rat intestine, liver
trichome cells. The stability of this molecule remains discussed and muscle tissue of abdomen and legs [6]. In another study, Romo
because it can give rise even in planta to several dehydrogenation Vaquero et al. [7] also investigated the bioavailability of CA and
derivatives such as carnosol, rosmanol and isorosmanol. In plants other diterpenoids found in rosemary extract and detected all the
subjected to intense solar radiation and high drought, CA may be metabolites 25 min after oral administration in rats. CA was
transformed into methyl derivatives. This clearly demonstrates pronouncedly present in the intestine, liver, and plasma with some
quantities of CA and its metabolites even in the brain, translating
their potential health benefits in these tissues.
Wang et al. [8] evaluated the acute and 30-day oral toxicity of
CA on Wistar rats and defined acute oral lethal dose (LD50) in the
range of 7100 mg/kg of body weight. In rats treated chronically
with a high-dose of CA, they observed slight reduction in the body
weight gain compared to control group and revealed only weak
pathological changes in the heart, liver, and kidney. These
observations reflect low toxicity profile of the molecule.

4. Anti-cancer effect of carnosic acid

4.1. Carnosic acid and colorectal cancer

The relevance of the current studies to the oncology is based on

the inhibition of the in vitro viability of different cell lines
Fig 1. Carnosic acid.
 S. Bahri et al. / Biomedicine & Pharmacotherapy 84 (2016) 569–582
originated from colon cancer. Barni et al. [9] initially studied CA
effect on 3 types of cultured colorectal cancer cells: Caco-2, HT29
and LoVo. They revealed a dose-dependent reduction in the
viability of these cells treated with CA with IC50 values within the
range of 24–96 mM. Cellular death followed the induction of
apoptosis and inhibition of cell migration and adhesion. This effect
was accompanied by a downregulation of the expression of COX-2
and by reduction of the activity of secreted proteases such as
urokinase plasminogen activator and metalloproteinases [9]. Kim
et al. [10] showed a decrease in the viability of human colon cancer
HCT116 cells treated with CA (20 to 100 mM). It triggered HCT116
cells apoptosis via ROS generation, p53 and Bax induction, caspase
3–9 activation, PARP cleavage and STAT3 signaling pathway
inhibition. The same Korean team demonstrated that CA could
slow down the rapidity of colon cancer formation associated with
adiposity. In fact, Kim et al. [11] discovered that treatment with CA
at 1 and 10 mM for 48 h attenuated the proliferation of HT-29 colon
adenocarcinoma cells co-cultured with 3T3-L1 adipocytes by
inducing cell cycle arrest and apoptosis. The in vitro molecular
mechanism involved the attenuation of leptin receptor signaling,
including downstreaminhibition of Akt and ERK phosphorylation.
Then, Kim and colleagues tested the effect of carnosic acid in a
mice model of colitis induced by treatment with dextran sodium
sulfate as well as in a model of colon cancer induced by
azoxymethane. Compared to untreated animals, mice treated with
CA have shown a reduction in the number of colon tumors and a
decrease in circulating concentrations of leptin, adiponectin,
insulin, and insulin-like growth factor 1. In addition, the expression
of leptin and insulin receptors, phosphorylated Akt and ERK, B-cell
lymphoma extra large (Bcl-xL), and cyclinD1 protein in the colon
were attenuated by CA treatment [11].
With regard to rosemary leaves extract, Borrás-Linares et al.
[12] reported its antiproliferative action on a group of human colon
cancer cell lines. The characterization and quantification of various
phenolic compounds in this extract by HPLC-ESI-QTOF-MS allowed
authors to point out the contribution of CA among other
compounds identified. Thus, they estimated IC50 value for carnosic
acid as 8.1 mM for SW 480 cells and 20.4 mM for HT29 cells.
In summary, all these findings cited above suggest that CA
might have clinical applications for the treatment of the colorectal
cancer, considered as the third most frequent malignant tumor
around the world [9].
4.2. Carnosic acid and liver cancer
The exploration of CA effect for the treatment of liver cancer is
not complete. Yet, current data clearly confirm the anticancer
activity of CA on tumor cell lines originated from rat or human liver
tumors. For example, Gao et al. [13] reported that CA caused an
autophagic cell death in human hepatoma cells (HepG2) but not
apoptosis, the latter being the major mechanism exploited by this
herbal molecule in other tumor cell lines. Data of this study
showed time and dose-dependent (within the range of 20–
100 mM) formation of autophagic vacuoles with an increase in the
ratio of cytosolic microtubule-associated protein 1A/1B-light chain
3 (LC3-I) and its conjugated form LC3-II with phosphatidyletha-
nolamine, used to detect autophagy-related processes. Further-
more, CA exerts this autophagic induction through the inhibition of
Akt/mTOR pathway [13].
In contrast, Xiang et al. [14] demonstrated that CA reduced cell
viability of HepG2 cells via induction of apoptosis in a dose-
dependent manner (5–100 mM),causing a rapid caspase-3 activa-
tion and a proteolytic cleavage of poly (ADP-ribose) polymerase
(PARP) accompanied by a mitochondrial dysfunction and deacti-
vation of Akt. Therefore, we should ask a question: how can one
explain the fact that CA can activate two different ways of cell
571
death in the same cell type? Common observation in these above
studies is that CA induced HepG2 cell death by Akt inhibition. This
leads us to conclude that Akt signaling suppression is implicated
both in apoptosis and autophagy in these cells. Most likely, slight
culture condition differences including different serum might
trigger HepG2 cells to undergo autophagy instead of apoptosis in
the presence of CA. Arico et al. [15] demonstrated that PI3 kinase/
Akt pathway activation, which is well known to inhibit apoptosis,
could also inhibit autophagy in human colon cancer HT-29 cells. In
addition, mTOR/Akt signaling pathway, death-associated protein
kinase (DAPK) and p53 activation were described to participate in
both processes [16].
The progress in molecular research on cell death demonstrates
that autophagy and apoptosis have a strong molecular conver-
gence. The discovery of the autophagy related genes (ATG) in
Saccharomyces cerevisiae, whichare mostly conserved from yeast to
human, has allowed a clarification of the mechanism controlling
this process. ATG genes have been identified to manage an orderly
process leading to the induction of autophagic vesicles, their
nucleation, completion, lysosomal fusion and finally their
recycling [17]. Several studies indicate that two types of ATG
can interact with Bcl-2 member family, FADD and induce
activation of caspases. In fact, ATG-5 can participate in the
formation of autophagosomes involving FADD death domain
binding [18]. Furthermore, ATG-12 can provoke Bcl-2 inhibition
and caspase activation [19–20,21]. Finally, Pattingre et al. [22]
demonstrated the close crosstalk between beclin-1 and Bcl-2 in
prevention of autophagy initiation. These examples provide the
evidence that some autophagy regulators have the ability to affect
both apoptosis and autophagy and prove the clear connection
between the machineries of these two processes. In some cases,
cell death in the presence of CA might be triggered by
simultaneous activation of both phenomenon involving Akt
inhibition. This reinforces effectiveness of CA as an anticancer
agent (Fig. 2).
On the other hand, pretreatment with CA can protect and
counteract the toxic effect of some carcinogenic agents. Costa et al.
[23] demonstrated a dose-dependent protective effect of carnosic
acid pretreatment on cell toxicity induced by aflatoxin B1 in HepG2
cells. When cells were pre-treated with relatively low doses of CA
(20 or 30 mM) for 24 h prior to aflatoxin, it decreased reactive
oxygen species level and cell death. This study highlighted the
preventive effect of this molecule, which can play a dual role as a
preventive and curative herbal drug against some type of cancer.
4.3. Carnosic acid and renal cancer
The studies of CA effect on human renal cancer started only in
the last two years by the Korean researchers Min and Jung [24].
Actual data are still limited to the detection of the in vitro effects in
human renal carcinoma Caki cells, but remain encouraging and
need to be continued.
CA induces apoptosis in human renal carcinoma Caki cells
incubated with 20, 40 and 60 mM for 24 h via caspase activation
associated with poly (ADP-ribose) polymerase (PARP) cleavage,
intracellular ROS production and endoplasmic reticulum (ER)
stress [24]. The induction of ER stress marker proteins expression,
the activation of transcription factor 4 (ATF4) and CCAAT/
enhancer-binding protein-homologous protein (CHOP), known
as a major ER stress-mediated pro-apoptotic transcription factor,
were also detected following CA treatment in a dose- and time-
dependent manner.
Jung et al. [25] demonstrated that CA at 10 or 20 mM induced
considerably Tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL)-mediated apoptosis in human renal carcinoma Caki
cells after 24 h of incubation through caspase-3- 8 and -9
572 S. Bahri et al. / Biomedicine & Pharmacotherapy 84 (2016) 569–582

Fig. 2. Possible connection between autophagy and apoptosis causing HepG2 cell death induced by carnosic acid.
Carnosic acid (CA)promotes the formation of autophagic vacuoles via ATG activation. The latter (probably ATG-12) can induce caspase activation and Bcl2 inhibition both
implicated in apoptosis. CA can also dissipate mitochondrial membrane potential provoking cytochrome c (cyt c) release, nuclear cleavage of PARP and BAX/Bcl 2 activation to
ensure apoptosis. These two processes implicate down regulation of AKT and may involve other molecular pathways separately to induce HepG2 cell death.

activation.TRAIL is known by its binding to death receptors (DR4
and DR5),whose activation leads to apoptosis in various cancer
cells. The investigation of the molecular targets and apoptosis-
related protein expression showed that CA at 20 mM sensitizes
TRAIL-mediated apoptosis through the down-regulation of anti-
apoptotic proteins such as cellular FLICE-inhibitory protein (c-FLIP)
and Bcl-2. This effect includes also an up-regulation of ER stress-
mediated death receptor5 (DR5), and up-regulation of the pro-
apoptotic Bcl-2 family proteins Bim and PUMA expression at the
transcriptional levels.
All these results demonstrate clearly that CA could be a potent
candidate for the treatment of renal cancer. An association with
other cell ligands like TRAIL can improve its action. Indeed,
combined action of CA and TRAIL can inhibit Caki cells viability
that is associated with chromatin damage in the nuclei and in
cytoplasmic histone-associated DNA fragments [24]. CA can
reduce the mitochondrial membrane potential and induce
cytochrome-crelease by the activation of the pro-apoptotic
protein (Bax) [24].
4.4. Carnosic acid and brain cancer
Studies of CA effect on human brain cancer remain scarce. The
first study on brain tumor cells has been performed by Kosaka et al.
[26], but the aim of this study was not to determine the anticancer
effect of CA, but rather to identify its role as a stimulator of nerve
growth factor (NGF) synthesis in human glioblastoma multiforme
T98G cells. De Olivera et al. [27] took advantage of SH-SY5Y
neuroblastoma cell model to determine the protective effect of CA
(1 mM for 12 h) against methylglyoxal cytotoxicity, found in
abundance in samples obtained from patients suffering Alzheimer
disease. The same research team studied the effect of CA in
paraquat model of Parkinson's disease using these cells, demon-
strating that this molecule can play a potential neuroprotective
role and can be used in some cases of Parkinson's disease [69]. The
antitumor action of CA in brain cancer was reported in vitro by Tsai
et al. [28]. It has been demonstratedthat CA can decrease the
viability of human neuroblastoma IMR-32 cells in a dose-
dependent manner (in the range of 5–40 mM). It induced apoptosis
associated with caspase-3/-9 and PARP activation and accompa-
nied by ROS generation and a down-regulation of anti-apoptotic
protein Bcl-2. The molecular investigation showed an increase of
p38 and JNK activation and a decrease in ERK activation [28]. We
conclude that further experimental studies are needed to
determine the anticarcinogenic effect of CA on several types of
brain cancer.
4.5. Carnosic acid and leukemia
Leukemia is a malignant progressive tumor characterized by
the formation of immature or abnormal leukocytes by the bone
marrow and other blood-forming organs leading to the deletion of
normal blood cells generation. The protective effect of CA against
leukemia was first described by Steiner et al. [5] in HL-60 and U937
human myeloid leukemia cells. This effect was explained by the
inhibition of cell proliferation by cell cycle arrest in the G1 phase
with an IC 50 in the range of 6–7 mM without inducing apoptotic or
necrotic cell death. Cell cycle arrest was evidenced from the
increase in the levels of the universal cyclin-dependent kinase
inhibitors p21WAFI and p27Kipl [5]. This anticarcinogenic activity
could be enhanced synergistically by 1,25-dihydroxyvitamin D3
and by all-trans retinoic acid(ATRA) at low concentration (1 nM).
CA at 10 mM cooperated with these two compounds to potentiate
their differentiating effects in human myeloid leukemia cells
[5,29]. To detect the mechanisms involved in this differentiation
enhancement, especially in sublime HL60G, Danilenko et al. [30]
demonstrated that this monocyte differentiation stimulation was
associated with an increase in monocytic serine esterase and
CD11b expression. The latter is a member of integrin family
expressed on the surface of many leukocytes that regulates their
adhesion and migration and contributes to the inflammatory
process. In addition, this combinatory effect of CA with 1a,25(OH)
2D3 and ATRA decreased cell proliferation and cell cycle transition
from G1 to S phase but increased Nuclear vitamin D receptor (VDR)
and Retinoid X receptor a (RXR-a) expression. Separately, CA at
10 mM and 1a,25(OH)2D3 at 1 nM induced a decrease in the ROS
intracellular level in HL60G cells. Added together, these molecules
ameliorated the antioxidant status, increased intracellular
 S. Bahri et al. / Biomedicine & Pharmacotherapy 84 (2016) 569–582
glutathione level andpotentiated monocytic differentiation via
mitogen-activated protein kinase pathway.
The effect of another analogue of vitamin D (1,25D(3)) was
investigated in vivo [31]. These authors demonstrated the
antileukemic efficacy of CA when combined with the hormonal
form of vitamin D 1,25D(3) analogue, 1,25-dihydroxy-21(3-
hydroxy-3-methyl-butyl)-19-nor-cholecalciferol, in a mouse mod-
el of acute myeloid leukemia. This team has performed a
subsequent study and focused on the combined effect of CA with
other plant polyphenols such as curcumin. They demonstrated that
these two polyphenols produced a synergistic antiproliferative
effect caused by an important apoptotic cell death in HL-60 and
KG-1a human acute myeloid leukemia cells, accompanied by
caspase 3–8 and-9 activation and this involved both extrinsic and
intrinsic apoptotic pathways. Curcumin at 5 mM and CA at 10 mM
can induce the activation of the proapoptotic protein Bid without
increasing ROS level or altering Bcl-2 family protein expression
[32].
On the other hand, this apoptotic response to curcumin and CA
in acute myeloid leukemia cells is associated with an elevation in
the cytosolic level of Ca2+ and with the inhibition of its extrusion
through the plasma membrane [33]. This Ca2+ release from
intracellular stores was found to be responsible for caspase cascade
activation. Interestingly, this combined treatment did not modify
Ca2+ homeostasis and viability in non-neoplastic hematopoietic
cells, indicating cancer-selective property.
CA also synergized with arsenic trioxide (As2O3), used as a drug
for cancer treatment, on HL-60 human myeloid leukemia cells that
has been described by Wang et al. [34]. Synergic decrease in cell
viability, the induction of G1 arrest and apoptosis associated with
the modulation of PTEN/Akt signaling pathway was observed in
the presence of both CA at 15 mM and As2O3 at 4 mM. Considering
all these results, we deduced that CA has a potent anti-tumor effect
against different cell lines of myeloid leukemia. This activity can be
boosted by other anticancer compounds, especially by plant-
derived polyphenols.
An important feature of cancer cells is the excessive production
of P-glycoprotein that has the ability to extrude anticarcinogenic
drugs from the cell providing it a multi-drug resistance (MDR)
phenotype. Identification of a natural molecule, capable to reverse
this phenotype in human leukemic cell line K562/A02 was the
main objective of Yu et al. [35] study. In this case, CA was found
capable to enhance the cytotoxicity of adriamycin, a chemotherapy
drug commonly used in cancer treatment, by increasing its
intracellular concentration. This reflected in a decrease of the IC50
from 16.3  1.6 mg/l when cells are treated with adriamycin alone
to 1.4  0.2 mg/l when cells are treated with adriamycin and CA. CA
was found to be able to decrease P-glycoprotein level and to
suppress MDRL gene expression at the mRNA level. These results
highlight the reversing effect of CA against multidrug resistance
mediated by P-glycoprotein in human leukemic cells.
Recent study by Wang et al. [36] of CA effect on NOD/SCID acute
myeloid leukemia mouse model established by K562/A02 cells
inoculation has been performed to verify if CA combined with
adriamycin can inhibit cancer growth in vivo. Data demonstrated
that mice treated with CA (1%, v/v) and adriamycin (3 intraperito-
neal injection of 1 mg/kg every two days) contained a lower
number of leukemia cells than untreated mice and a higher level of
apoptotic cells than in adriamycin-alone treated mice, which
demonstrates that CA has an important inhibitory activity on the
malignant cells growth in vivo.
Taken together, current data reveal the antitumor property of
CA in leukemic cell lines, which can be summarized in two major
actions: 1) its ability to cooperate with other anticancer agents and
2) its reversing effect on MDR phenotype. Others studies are
required to determine if this cooperation can ameliorate CA MDR
573
reversing activity and to identify these properties in various cancer
cell lines.
4.6. Others anticancer properties of carnosic acid
CA has other therapeutic virtues by killing or reducing the
growth of various cancer cell lines and by the treatment of
angiogenesis-related malignancies. Indeed, angiogenesisis is a
physiological process of neovascularisation from the pre-existing
blood vessels occurring particularly during embryonic develop-
ment. This process appears to be also important for the growth of
malignant tumors and metastasis. Then an anti-angiogenesis
treatment can prevent the formation of new blood vessels and
block the progression and the growth of tumors. CA at 10 mM and
100 mM has been described to have an anti-angiogenic activity by
inhibiting differentiation, proliferation, migration and proteolytic
capability of endothelial cells and by exerting an antiproliferative
effect in endothelial and tumor cells through induction of
apoptosis [37]. The use of chicken embryo chorioallantoic
membrane assay as a model for studying neovascularisation
confirmed the inhibition of both in vitro and in vivo angiogenesis
process described above. CA was described also to suppress
angiogenesis in models using human umbilical vein endothelial
cells and to inhibit microvessel outgrowth on ex vivo angiogenesis
test using rat aortic rings in a dose-dependent manner (10–
100 mM) [38]. This activity may contribute to the prevention of
angiogenesis-related disorders including cancer and neurodegen-
erative diseases.
Melanoma is the most serious skin cancer type that develops
from melanocytes that provide a pigment for skin coloration
named melanin.CAwas shown to prevent murine B16-F10 mela-
noma cells adhesion and migration at concentrations ranging from
2.5 to 10 mM via the suppression of the mesenchymal marker
expression and via induction of epithelial markers leading to the
inhibition of the epithelial-mesenchymal transition (EMT) with an
inhibition of AKT phosphorylation among other signaling path-
ways involved [39].
CA could be proposed and taken into consideration for the
prevention and/or for the treatment of prostate cancer, but actual
results remain insufficient and need to be more elaborated. In fact,
CA at concentrations of 20–100 mM has anti-proliferative effects
on androgen-independent human prostate cancer PC-3 cells and in
androgen refractory prostate cancer DU145 cells caused by
apoptosis that implicates cytochrome-c release and an increase
in Bax/Bcl-2 ratio [40]. In PC-3 cells, these effects involve both
intrinsic and extrinsic apoptotic pathways as well as suppression of
the expression of inhibitor of apoptosis (IAP) family of proteins via
the activation of serine/threonine protein phosphatase 2A (PP2A)
and the modulation of Akt/IKK/NF-kB pathway. In DU145 cells, CA
induced apoptosis via the intrinsic pathway associated with PARP
cleavage. This study remains the only one that investigated the
efficacy of CA to treat human prostate carcinoma. Other studies are
required to better understand its mechanism.
CA was described to exert higher antiproliferative activity
compared to the effect of six different extracts of Rosmarinus
officinalis and rosmarinic acid in two human breast adenocarcino-
ma cell lines, MCF-7 and MDA-MB-231 [41]. In addition, CA
suppressed proliferation, induced G1 cell cycle arrest and
displayed significant growth inhibitory activity when associated
with curcumin in ER-negative human breast cancer cells [42]. In
fact, it was reported that treatment of these cells with CA for 6 h at
low concentration (15 mM) activated the genes involved in
glutathione biosynthesis and transport. At a higher dose
(20 mM), CA activated the expression of antioxidant and apoptosis
genes and suppressed the expression of inhibitors of transcription
and cell cycle genes.
574 S. Bahri et al. / Biomedicine & Pharmacotherapy 84 (2016) 569–582
Taken together, these results are a good start on the way to the
broad use of CA in the prevention or eradication of several forms of
cancer. Generally, this molecule appears to exert its antitumor
action by inhibiting cell viability via induction of apoptosis.
However, most of the studies did not go beyond the in vitro
experimentation. This constitutes the biggest limitation. Further
research and preclinical studies are required to fully elucidate and
to understand the potential of CA as a preventive or therapeutic
agent against various human carcinomas.
5. Carnosic acid and experimental disorders
A number of studies performed over the last decade up to date
confirm that CA has beneficial effects in the majority of models of
experimental diseases. In this section, we will recapitulate and
discuss the different data published in this regard.
5.1. Carnosic acid and liver injuries
Examination of CA beneficial effect in liver lesions induced by
various toxic agents advanced in the last three years. They
highlight specifically its antioxidant and anti-inflammatory
activities in several experimental studies.
CA has been studied on lipopolysaccharide [43], ethanol [44]
and acetaminophen [45] induced models of hepatotoxicity in rats.
CA normalized the majority of pathological signs including
histological damage, lipid metabolism and oxidative/nitrosative
stress by restoring the antioxidant enzymes levels both in serum
and liver and by reducing the inflammatory cells infiltration in liver
tissues. Moreover, several in vivo and in vitro reports demonstrated
that the molecular mechanism implicated in the protective effect
of CA against liver injuries involves sirtuin-1 (SIRT-1), an NAD-
dependent deacetylase that plays an important role in metabolic
diseases, such as nonalcoholic fatty liver disease (NAFLD) [46]. For
example, Shan et al. [46] suggested that miR-34a, that targets
SIRT1 expression, might be decreased in response to CA
administration in NAFLD rat model at a dose of 60 mg/kg. This
in turn stimulated the SIRT1/p66shc antiapoptotic signaling
pathway in rat liver and L02 cells. Tian et al. [44] reported that
10 mM CA pretreatment increased SIRT1 expression following
ethanol exposure in L02 cells, but the p66Shc expression and
mitochondrial translocation were negatively correlated with SIRT1
expression. Gao et al. [47] demonstrated that CA administered at a
dose of 100 mg/kg protected from chronic alcoholic liver injury by
activating the SIRT1/ChREBP and SIRT1/p66shc pathways.
Other in vitro experiments emphasizing the cytotoxicity of
hydrogen peroxide (H2O2) and the protective effect of CA at low
concentration (1 mM), on primary hepatocytes and HepG2 cells
have also reported the implication of SIRT1 [48,49]. Indeed,
pharmacological inhibition and the use of siRNA mediated SIRT1
silencing confirmed the activation of SIRT1 by CA in these two cell
types. CA was also reported to reverse the increase in ROS level,
LDH release and the decrease in cell viability produced by H2O2.
There is now enough experimental evidence that CA can attenuate
acute liver lesion and hepatotoxicity especially by alleviating
oxidative stress through the SIRT1 pathway. A parallel activation of
other signaling pathways may occur depending on the cell type
and on the molecule inducing liver damage.
5.2. Carnosic acid and cardiac injuries
Until now, only two published experimental studies focused on
the effect of CA in the context of cardiovascular disorders. They
made use of isoproterenol, a beta-agonist drug, to induce
myocardial injury in mice and rats. This drug can induce
histopathological lesions and inflammation and to increase serum
levels of several markers of myocardial infarction and lesion,
including troponin, myocardial creatine kinase (CK-MB), lactate
dehydrogenase (LDH), aspartate-aminotransferase (ASAT) and
alanin-aminotransferase (ALAT). CA pretreatment at 50 or
100 mg/kg can attenuate all these alterations; it also prevented
cardiomyocyte apoptosis and decreased myocardial lipid peroxi-
dation and protein oxidation induced by isoproterenol. This occurs
via the enhancement of nuclear translocation of Nrf2 and via an
up-regulation of the phase II/antioxidant enzyme activities [50].
Recently, Kocak et al. [51] confirmed all these biochemical results
and reported that in addition to the increase of Nrf2 expression, CA
at 50 mg/kg reduced TNF-a levels, NF-kB, p38 MAPK, and
phosphorylated-JNK 1/2 expressions, and enhanced phosphory-
lated extracellular signal-regulated kinase (pERK 1/2). In conclu-
sion, the reduction of myocardial impairment and apoptosis is
mediated by the antioxidant and anti-inflammatory action of CA.
5.3. Carnosic acid and atherosclerosis
Atherosclerosis is very often accompanied by lipid deposition
(cholesterol or LDL) on the inner wall of the arteries. Over the years,
deposits progressively impregnate with fibrinogen, platelets, blood
cells, calcium and solidify, thus blocking the arteries. According to
Yu et al. [52], migration and matrix metalloproteinase (MMP)
activation in vascular smooth muscle cells could participate in
atherosclerosis evolution and formation. In this regard, they
demonstrated that CA at 20 mM inhibited TNF-a-induced migra-
tion, ROS production, MPP-9 expression, and down regulated
nuclear translocation of NF-kB subunits p50 and p65 in human
aortic smooth muscle cells. The inhibition of nuclear translocation
of NF-kB subunits p65 and p50 was also reported along the
demonstration of CA effect on the expression of cell adhesion
molecules and on the attachment of monocytes to the endothelium
[53]. These authors showed that pretreatment with CA at 20 mM
reduced the U937 cells adhesion to IL-1beta-treated human
umbilical vein endothelial cells (HUVECs), suppressed ROS
production and the expression of cell adhesion molecules
(ICAM-1, VCAM-1, and E-selectin) via the inhibition of NF-kB.
In conclusion, we have limited experimental data to assess the
effectiveness of CA in human atherosclerosis. However, the
published studies are encouraging and need to be associated with
other clinical trials to better understand this effect on several
pathophysiological signs and clinical manifestations that charac-
terize this disease.
5.4. Carnosic acid and brain injuries
5.4.1. Carnosic acid and Alzheimer disease
Alzheimer is a degenerative disease that causes progressive
decline of cognitive capacity and memory that emerges especially
due to excessive production and accumulation of beta-amyloid
proteins in some areas of the brain. This forms amyloid plaques
that are associated with neuronal death and the “distortion” of
certain structural proteins, called Tau proteins, causing a neurofi-
brillary degeneration.a- and g-secretases can counteract beta-
amyloid formation resulted from the sequential cleavage of its
precursor, amyloid precursor protein (APP) by the b- and
g-secretases [54]. The pathological mechanism of this disease
implicates also oxidative stress and inflammation.
Azad et al. [55] reported for the first time the beneficial effect of
CAin experimental rat model of Alzheimer disease. They demon-
strated that carnosic acid at a dose of 3 mg/kg protected
hippocampal neurons from cellular death, especially in the cornu
ammonis 1 (CA1) region of the hippocampus, and this in turn
prevented beta-amyloid-induced neurodegeneration. Rasoolijazi
et al. [56] confirmed these results and explored the effect of CA,
 S. Bahri et al. / Biomedicine & Pharmacotherapy 84 (2016) 569–582
administered at the same dose of 3 mg/kg, on memory and
behavioral alteration caused by beta-amyloid. They demonstrated
that this herbal molecule could ameliorate the spatial and learning
memory deficits. To better understand this mechanism, Meng et al.
[57] evaluated the effect of CA at 30 mM on beta-amyloid
production in SH-SY5Y human neuroblastoma cells and found a
reduction of its secretion via tumor necrosis factor-a-converting
enzyme activation (a-secretase TACE) without effect on the b-site
APP-cleaving enzyme-1 (b-secretase, BACE1). Later, the same
research team demonstrated that beta-amyloid treatment induced
SH-SY5Y cells apoptosis by the cleavage of PARP and by the
activation of caspase 3–8 and 9, while CA pretreatment at 10 mM
induced a partial inhibition of apoptosis and reduced beta-amyloid
oligomer cellular content [58]. Further studies are required to
elucidate the signaling pathway involved in this protective effect of
CA. However, it is interesting to note that, in SH-SY5Y cells, CA
pretreatment (1 mM for 12 h) can counteract methylglyoxal
neurotoxicity by activation of the PI3K/Akt/Nrf2 signaling pathway
and by up-regulation of Nrf2-dependent antioxidant enzymes [59].
In this experiment, methylglyoxal was used as a potent inducer of
glycation. Methylglyoxal seems to be a key molecule in case of
neurodegenerative diseases such as Alzheimer, because samples
obtained from subjects suffering Alzheimer contain high amount
of methylglyoxal. However, it remains important to clarify whether
CA neuroprotection against beta-amyloid neurotoxicity directly
inhibits glycation or whether it requires the participation of other
signaling molecules.
5.4.2. Carnosic acid and Parkinson disease
The development and characterization of relevant in vitro/in
vivo models of Parkinson disease using several neurotoxins seems
to be a practical tool to study the capacity of certain drugs and food
constituent to counteract or prevent many deleterious effects
accompanying this disease. These neurotoxins include dieldrin, an
organochlorine insecticide involved in sporadic Parkinson's
disease development [60]; 6-hydroxydopamine (6-OHDA), a
neurotoxic organic compound used to kill selectively dopaminer-
gic and noradrenergicneurons that constitutes the most commonly
used drug for the elaboration of animal model of Parkinson's
disease [61]; cyanide, a toxic chemical compound inducing
delayed-onset neurological syndrome with the symptoms of
Parkinsonism [62]; and paraquat, a toxic herbicide used for the
establishment of Parkinson experimental model [63]. All these
neurotoxins were used by various research teams worldwide to
provide new insights on different mechanisms involved in CA
induced neuroprotection.
To evaluate the effect of various neurotoxins and create an in
vitro/in vivo model of Parkinson disease, several neuronal cells
types/animal models were used. For example, Park et al. [64]
examined the effect of CA pretreatment (at 5 or 10 mM) on dieldrin
induced-neurotoxicity in cultured dopaminergic cells (SN4741)
and showed a dose-dependent protective effect. Chen et al. [65]
demonstrated that CA attenuated SH-SY5Y cell death induced by 6-
OHDA in a dose-dependent manner within the range of 0.1–1 mM.
Two years later, Lin et al. [66] explored the contribution of
glutathione S-transferase (GST)-P in this neuroprotective effect in
the same cell type and used 6-OHDA-treated rats to demonstrate
an improvement of the locomotor activity after 1 mM CA
pretreatment. Later, they revealed that the reduction in 6-
OHDA-induced SH-SY5Y cell death by CA at the same dose was
associated with the induction of parkin, a Parkinson disease linked
gene that in turn up-regulated the ubiquitin-proteasome system
[67].
Zhang et al. [68] demonstrated a potent neuroprotective effect
of CA at 10 mM against cyanide brain poisoning, both in vitro using
cultured human and rodent pluripotent neuronal stem cells and in
575
vivo using cyanide-stimulated non-Swiss albino mouse brain areas
which show several deteriorations similarly to what has been
detected in human brain. Finally, de Olivera et al. [69] investigated
the role of CA on SH-SY5Y cells treated by paraquat to create an
experimental model of Parkinson disease.
The activation of the nuclear factor erythroid-2 related factor 2
(Nrf2) plays an important role in the neuroprotective effect of CA in
both in vitro and in vivo models of Parkinsonism induced by several
neurotoxin [65,68,69]. This effect may be accompanied by the
inhibition of JNK and p38 [64–65,70] or by the modulation of PI3K/
Akt pathways [67,69]. CA exerted also an antioxidant action by
suppressing ROS [65] and RNS [66] production, by the increase of
total glutathione level (GSH) [65,67] and of glutathione S-
transferase activity (GST)-P [66], and by the prevention of lipid
peroxidation [64]. CA has been described to reverse caspase
activation [64–65,70] and peroxisome proliferation-activated
receptor gamma (PARPg) cleavage [65,70] induced by several
neurotoxins.
All these results suggest that CA treatment might be a
promising management of Parkinson's disease.
5.4.3. Carnosic acid and retinal degeneration
CA may have clinical applications for different diseases
affecting the outer retina. Rezaie et al. [71] demonstrated that in
retina-derived cell lines ARPE-19 and 661W treated with hydrogen
peroxide, CA at 10 mM triggered the production of antioxidant-
phase 2 enzymes and was found to reduce the formation of
hyperoxidized peroxiredoxin (Prx2). CA was tested then in rat
model of light-induced retinal degeneration. Compared to
untreated animals, rats that were given CA (25 mg/kg/day, for
6 days) had a significant improvement in outer nuclear layer
thickness and a better electroretinography activity. This study
confirmed that CA might have clinical application to protect the
retina and other areas of the brain from degenerative disorders,
including age-related macular degeneration and retinitis pigmen-
tosa. The latter is a hereditary retinal disease with progressive
degeneration of photoreceptors (produced following rod-specific
gene mutations) leading to partial or complete vision loss.
Recently, Kang et al. [72] reported that CA preserved photo-
receptors morphology and function in the Pde6b (rd10) mouse
model of retinal pigmentosa, through inhibition of oxidative stress,
endoplasmic reticulum stress and inflammation, and by the
regulation of multiple cellular pathways in mice including an
upregulation of Nrf2 expression and a downregulation of p-JNK
and p-p38 expression among others. These data provide the
evidence that CA could exert neuroprotective effects by defending
brain cells from oxidative stress, known by its implication in the
progression of several retinal diseases.
5.4.4. Carnosic acid protects from other neuronal agressions
CA is a catechol ring containing compounds that can be
converted by oxidation to an electrophilic quinone, characterized
by its ability to react with specific cysteine residues on targeted
proteins via thiol (S-) alkylation. The Kelch ECH associating protein
1 (Keap1) Nrf2 pathway is the major regulator of cytoprotective
responses to endogenous and exogenous stresses caused by
reactive oxygen species and electrophiles [73].
The involvement of (Keap1-Nrf2) pathway in the neuro-
protective effect of CA against oxidative stress and excitotoxicity
was first described by Satoh et al. [74] in cerebrocortical neurons.
They demonstrated that this activation was initiated by S-
alkylation of the Keap1 protein critical cysteine thiol by the
“electrophilic” quinone-type of CA. Then, Satoh et al. [75]
suggested that the protective effect of CA at 10 mM against
oxidative glutamate toxicity on HT22 cells required both free
carboxylic acid and catechol hydroxyl moieties to exert a
576 S. Bahri et al. / Biomedicine & Pharmacotherapy 84 (2016) 569–582
regulatory action on cellular redox impairment. This occurs via
activation of the transcriptional antioxidant-responsive element of
phase-2 genes, including gamma-glutamyl cysteine ligase, hem-
eoxygenase-1 and NADPH-dependent quinone oxidoreductase,
accompanied by an upregulation of glutathione expression and
metabolism [76].
Nrf2 regulates the expression of antioxidant and cytoprotective
proteins, and that requires its binding to the antioxidant-response
element (ARE) within DNA [77]. Several studies cited above
demonstrated that Nrf2 can be activated by CA to exert its
cytoprotective effect. Indeed, a prior administration of CA at 1 mg/
kg suppressed the inhibition of mitochondrial respiration induced
by 4-hydroxynonenal (4-HNE)-treatment in rat cerebral cortical
cells cultured ex vivo via the reduction of the level of 4-HNE bound
to mitochondrial proteins and via Nrf2-ARE activation [77]. This
signaling pathway is also implicated in CA neuroprotection from
mitochondrial oxidative damage after experimental traumatic
brain injury in mice [78]. CA administered after injury at a dose of
3 mg/kg maintained mitochondrial respiratory function and
reduced cytoskeletal breakdown, lipid peroxidation and protein
nitration via Nrf2-ARE activation.
It was also reported that carnosic acid at 15 mM favors neurite
outgrowth and neuronal differentiation of PC12h cells via Nrf2-
p62/ZIP pathway, which enhances the cell surface TrkA neuro-
trophin receptor signaling, known to be implicated in neuronal
survival and differentiation by several neurotrophin such as NGF
[79]. The latter could be also activated by CA via Nrf2 pathway in
human T98G glioblastoma cells and normal astrocytes. NGF-gene
induction can be produced by CA in a dose/time dependent manner
(up to 50 mM, incubated for 2 h or 24 h) in parallel with an increase
in Nrf2 expression as well as that of its target genes [80]. Vaka et al.
[81] confirmed these results in vivo and demonstrated that
intranasal delivery of CA at a dose of 4 mg/kg together with
chitosan can enhance the endogenous levels of neurotrophins
including NGF. This suggests that CA is potential herbal molecule
that can treat body alterations associated with neurotrophins
shortage.
In summary, Nrf2 pathway appears to be an important
molecular effector following CA treatment in many experimental
studies (Fig. 3). Nonetheless, other signaling pathways may be also
involved. For example, CA exhibits anti-nociceptive property in
Fig. 3. Alleged involvement of Nuclear factor erythroid-2 related factor -2 (Nrf 2) in the protective effect of carnosic acid against brain disorders.
Carnosic acid (CA) inhibits methylglyoxal-induced glycation increase in Alzheimer brain via activation of Nrf2/PI3K/AKT signaling pathway. CA preserved photoreceptors
morphology and function in an experimental model of retinal pigmentosa and attenuated experimental model of parkinsonism induced by 6-OHDA, Paraquat or Cyanide by
an up-regulation of Nrf2 and a down-regulation of JNK/p38.
neuropathic pain model via spinal sirtuin1 activation and down-
regulation of p66shc expression [82]. In conclusion, Nrf2 pathway
can be a common molecular mechanism for neurotrophic
properties of CA, while tissue-specific neuroprotective action of
CA may involve also other effectors.
5.5. Canosic acid and obesity
Obesity is a serious health disorder caused by accumulation of
excessive amount of body fat that may increase the risk of other
diseases such as diabetes, arthritis, heart troubles and cancer.
Overweight develops following high-fat diet, lack of physical
activities or may be genetically related. Treatments that limit
adipogenesis progression and differentiation can be a good
strategy to cure obesity-related diseases. Thus, some studies have
elucidated the antiadipogenic effects of CA (at a dose ranging from
0.1 to 10 mM). These studies have shown an activation of the
antioxidant-response element (ARE), the induction of phase2
enzymes and an up-regulation of intracellular level of total GSH
[83].
CA treatment on the first day of differentiation of preadipocytes
3T3-L1 into adipocytes can inhibit adipogenesis, associated with a
suppression of mitotic clonal expansion and with expression
inhibition of critical transcriptional factors of adipogenesis, C/
EBPa and PPARg [84]. This antiadipogenic mechanism involves
also an attenuation of lipid accumulation in 3T3-L1 adipocytes [85]
and HepG2 cells [86] with a down-regulation of triglycerides level
accumulated in adipocytes especially at CA concentration of 10 mM
[85]. CA was described also to repress palmitate-induced cellular
lipid accumulation in vivo via activation of EGFR and MAPK
phosphorylation [86]. However, it should be noted that the use of a
nontoxic dose of CA was essential. In fact, Dickmann et al. [87]
showed that CA exhibits a dose-dependent hepatotoxicity in
primary human hepatocytes with an IC50 value of 94.8  36.7 mM.
It could still increase activity of some members of cytochrome
P450 superfamily enzymes in human liver microsomes when
administered at 10 mM. These results provide an indication of a
safe dose of CA to avoid its toxicity and to obtain a beneficial
pharmacological effect for the induction of weight loss.
Various experimental studies have used rosemary extract rich
in CA to demonstrate its preventive/curative effect in obesity and
 S. Bahri et al. / Biomedicine & Pharmacotherapy 84 (2016) 569–582
to confirm its reducing effects on body weight gain without
modification of food intake [88–92,93]. This mechanism was
further elucidated by the evaluation of several biochemical
markers, such as glycemia, plasma cholesterol level, serum/liver
triglycerides, insulin level, plasma ALT and AST and free fatty acid
levels, which were reduced by the administration of rosemary
extract compared with control diet. This resulted from the
inhibition of gastric lipase activity [90] and was accompanied by
an improvement in glucose tolerance [92].
The antioxidant power of rosemary extract containing 80% of
CA was evidenced by a reduction of malondialdehyde levels in
plasma and liver and by an increase in the GSH/GSSG ratio in the
liver [93]. The anti-inflammatory effect was revealed by a
reduction in circulating IL-1b, leptin, adiponectin and tumor
necrosis factor alpha levels when rats are treated with rosemary
extract containing 40% of CA [91]. In addition, the high amount of
fecal lipid excretion was behind the suppression of lipid
absorption and the diminution of lipid accumulation in mouse
hepatocytes [93]. The decrease in body weight gain was
attributed also to the modulation of microbiota composition
and a reduction of b-glucosidase content in rat caecum with an
increase in its fecal evacuation [94]. The molecular mechanisms
accompanying these hepatic regulations by rosemary extract
implicate a reduction of phase I and phase II gene expression [91],
an activation of the expression of receptor of advanced glycation
end products (RAGE) [93] and activations of hepatic lipogenic-
related genes with an increase in lipolysis-related genes
expression [92].
The majority of experimental studies designed to evaluate the
antiobesity effect of rosemary extract demonstrated that this
molecule can be a promising agent to attenuate metabolic
disorders by regulating fatty acid metabolism, hepatic fat
accumulation, glucose tolerance and various other manifestations.
5.6. Carnosic acid and gastric lesions
Doolaege et al. [6] demonstrated that CA given in a single dose
orally (64.3  5.8 mg/kg) is absorbed in rats into blood circulation.
After this oral administration as well as after single intravenous
dose (20.5  4.2 mg/kg) traces of CA were located in gut, abdomen,
liver, legs and muscle tissues. The distribution of CA in rats after
intragastric administration (90 mg/kg) is limited to stomach and
intestine [95] and the molecule is eliminated through the fecal
route. To assess its gastroprotective activity, some studies
compared the effect of different CA derivates in vivo using HCl/
EtOH-induced gastric lesions model in mice or rat and in vitro on
different gastric cell lines.
In fact, the gastroprotective action of a single oral dose of CA
alkyl derivatives (40 mg/kg) has been demonstrated to correlate
with the length of the alkyl chain in the molecule and the best
effect was attributed to oleate and palmitate derivates [96].
According to Theoduloz et al. [97], some derivatives given at 10 mM
show a better preventive action with low cytotoxicity against
gastric lesions compared to the reference compound lansopra-
zole at the same dose. The elucidation of processes involved in this
gastroprotective effect was performed using human cell culture
models, such as human gastric adenocarcinoma cell line (AGS) and
lung fibroblasts (MRC-5) treated with sodium taurocholate (NaT)
that produces cell lesions [97]. Gastroprotective properties of CA
included an increase in the cellular reduced glutathione (GSH) and
prostaglandin E2 (PGE2) levels, protection against cell lesion
induced by NaT and inhibition of lipid peroxidation. However, the
signaling pathways behind these effects are still imperfectly
understood. Further studies using these gastroprotective mole-
cules derived from CA are required to determine the molecular
mechanisms involved.
577
5.7. Carnosic acid and skin lesions
Ultraviolet (UV) radiation is able to penetrate deeper into the
skin to provoke premature dermal ageing characterized by a
gradual loss of elasticity and by the development of dry and rough
skin. UV is known to induce oxidative DNA damage by ROS
production and to activate various types of matrix metalloprotei-
nase (MMPs) in the skin. This implicates epidermal growth factor
receptor (EGFR) and extracellular signal-regulated kinase (ERK)-
mediated AP-1 signaling [98,99].
CA at low concentrations ranging from 0.3 to3 mM has been first
reported to suppress the increase of metalloproteinase 1 (MMP-1)
mRNA level in human skin fibroblasts exposed to UVA radiation
[100]. Park et al. [101] confirmed this dose-dependent inhibitory
effect of carnosic acid against UVA/UVB induced MMP-1, MMP-3
and MMP-9 expression in a dose–dependent manner (3–10 mM) in
human dermal fibroblast cell line (Hs68), primary normal human
dermal fibroblasts (HDFs) and normal human epidermal kerati-
nocytes (HEKs). CA prevented ERK and AP-1 activation and reduced
ROS level induced by UV [101].
CA at 20 mM can also exert an anti-inflammatory action in skin
by attenuating several interleukins and chemoattractant protein
production, and by inhibiting tumor necrosis factor TNF-a,
prostaglandin E2 (PGE2) and nitric oxide (NO) release following
an inflammatory reaction [102]. This may be mediated through the
blockade of the nuclear translocation of nuclear factor (NF)-kB and
through the inhibition of Src-family/Syk tyrosine kinases.
5.8. Other possible therapeutic applications of carnosic acid
Various publications have shown others properties of CA. Lee
et al., [103] reported the antiplatelet activity due in part to the
blockage of collagen, arachidonic acid, and thrombin-mediated
cytosolic calcium mobilization with IC50 values of 39  0.3 mM,
34  1.8 mM and 48  2.9 mM. CA at 60 mM and 150 mM can exert
an antiviral activity against human respiratory syncytial virus
(hRSV) infection by suppressing its replication, inhibiting both A
and B type hRSV and blocking their genes expression [104]. CA at
100 mM can also attenuate cisplatin-induced experimental neph-
rotoxicity due to its strong antioxidant and antiapoptotic proper-
ties, through the inhibition of lipid peroxidation, caspase-3 and
ROS production and through the enhancement of some antioxidant
enzymes levels [105]. Other beneficial property have been
identified to be the counteraction of obesity-associated hypergly-
cemia and insulin resistance, by the improvement of glucose
homeostasis and by the amelioration of peripheral glucose
clearance in skeletal muscle tissue (L6 myotubes) through a
PME-1/PP2A/PKB signaling pathway when CA is administered at
20 mM [106]. Similar results were observed in vitro, where CA at
10 mM attenuated insulin resistance in 3T3-L1 adipocytes via Akt-
dependent FoxO1 signaling pathway [107]. Likewise, CA can exert
an anti-inflammatory action against tumor necrosis factor-a (TNF-
a)-inducing mRNA expression of inflammatory genes in 3T3-L1
adipocytes via NF-kB and AP-1 pathways.
These studies suggest that CA may regulate several cellular
alterations and target different molecular/metabolic mediators in a
tissue-specific manner, especially depending on the type of the
lesion occurred.
6. Carnosic acid synergizes with other natural compounds
We have mentioned above that the antileukemic action of CA
was boosted by a combinatory treatment with natural compounds
such as 1a,25(OH)2D3 vitamin D derivate, retinoic acid and
curcumin. CA also enhanced arsenic trioxide-induced HL60 human
myeloid leukemia cells apoptosis. This synergizing property of CA
578 S. Bahri et al. / Biomedicine & Pharmacotherapy 84 (2016) 569–582
has been demonstrated from the beginning of the experiments
designed to explore the anti-carcinogenic effects of this molecule.
Fuhrman et al. [108] proved also that tomato lycopene (5 mM)
acts synergistically with CA (25 mM), as an effective antioxidant
against low-density lipoprotein (LDL) oxidation, known to cause
atherosclerosis. Lycopene belongs to the family of carotenoids. It is
a natural pigment that gives red color to plants such as tomato.
Insoluble in water, lycopene is transported by the blood lipids and
is accumulated in certain organs, particularly in the liver and
prostate. This study showed that lycopene exhibited synergistic
antioxidative effect with other components such as vitamin E,
glabridin, rosmarinic acid and garlic against LDL oxidation, induced
either by copper ions or by the radical generator AAPH. This
indicates that the molecular recombination phenomenon can
occur with this natural compound and is not specific to CA.
Another original recent study aimed to develop CA derivates in
silico able to inhibit acetylcholinesterase activity, in order to
reverse the lack in acetylcholine content that causes memory
damage [109]. In this context, CA derivates cooperate with
acetylcholinesterase to induce a negative feedback, i.e. down
regulation in its expression. This study offers an alternative
strategy to treat Alzheimer's disease.
In summary, synergistic action of CA with other molecules may
constitute the basis of a combinatory treatment approach against
the emergence of cancer cells, but also against other dysfunctions
that may occur in the human organism. It is important to figure out
whether the synergy occurs at the level of cellular signalization or
the chemical combination takes place in the molecule that creates
new chemical compounds responsible of the enhancement of
several specific properties of CA.
7. Rosmarinic acid and carnosic acid: possible connection
CA has been described to produce a synergistic beneficial effect
in human diseases by cooperating with other natural compounds,
such as 25-dihydroxyvitamin D3, all-trans retinoic acid and
curcumin. Cooperation between CA and other phenol compounds
in planta such as rosmarinic acid is very possible to boost its
biological activities. This reinforces the power of rosemary extract
to treat several human diseases. Indeed, rosmarinic acid (RA,
C18H16O8, Fig. 4) is one of the most important component of
rosemary and it been isolated from many species of the Lamiaceae
family [110]. RA and its derivatives are easily produced in cell
cultures in high yield without a special handling, because this
substance is one of the secondary metabolites that are synthesized
continuously in plants [111].
Many biological activities of RA have been reported. These are
mainly the anti-inflammatory [112], anti-angiogenic [113], anti-
oxidant [114], anti-apoptotic [115], and anti-fibrotic [116] activi-
ties. These properties could be potentiated in cooperation with CA.
In this context, our research team demonstrated synergic potential
between CA and RA in human lung fibroblasts, rat lung fibroblasts,
rat alveolar type II cells and L929 cells cultured in vitro (submitted).
We reported an enhancement in the proapoptotic activity of CA in
these cells in the presence of RA, while RA alone did not
Fig. 4. Rosmarinic acid.
compromise cell viability. These results highlight the strong ability
of RA to bind and increase the effects of CA.
8. Conclusion
Numerous evidences of the beneficial effect of CA for the
treatment of various health disorders have been published.
Related mechanisms include the inhibition of oxidative stress, the
attenuation of inflammation and cancer. In the latter case, CA can
attenuate tumor progression via apoptosis induced by ROS
generation and caspase activation. This implicates in some cases
both intrinsic and extrinsic apoptotic pathways, ER stress
induction and PPAR cleavage [33]. The molecular pathways
involved in this preventive/therapeutic effect of CA against some
type of cancer are summarized in Table 1. It is important to
point out that there is no particular or universal signaling
pathway taken by CA for the killing of different types of cancer
cells. Indeed, CA on various types of carcinomas may induce
apoptosis, cell cycle arrest, autophagy, and inhibition of cellular
proliferation Table 1. In each case, different signaling pathways
are involved. In some cases, even opposite modulation of a
signaling pathway is reported; as can be seen, Akt is activated in
leukemia cell, while it is rather down-regulated in other tissues in
the presence of CA.
Great variability of modulation of signaling pathways is
observed in the treatment of other disorders besides cancer. As
shown in Table 2, effects of CA can be arbitrary divided into three
groups. First group of effects concerns cell viability. Major
pathways modulated by CA are sirtuin-1, PI3K/Akt, Keap-1/Nrf2,
MAPK-p38/JNK and ERK1/2. One can witness cell specific action of
CA for MAPK-p38/JNK pathway. While in neuronal cells this
pathway is activated to protect against apoptosis [70], MAPK-p38/
JNK is inhibited by CA to produce antiapoptotic effect in myocytes
[51]. However, it is difficult to compare these two studies because
of uncertainty about the in situ concentration of CA in the study of
Kocak et al. [51]. The dose used in this study (50 mg/kg) is rather
high and, given the high bioavailability of CA, this could be
translated into higher micromolar concentration of CA in the
myocardium. Thus, we could suggest concentration dependent
dual effect of CA on MAPK-p38/JNK pathway. Better example of
dose-dependent signaling pathway modulation is the inhibition of
PI3K/Akt in cancer cells by high-micromolar CA concentrations and
PI3K/Akt activation in neuronal cells by low-micromolar concen-
trations (Tables 1 and 2). Similarly, in comparison with the Table 1,
one can note that, at low micromolar concentrations, carnosic acid
induces rather anti-apoptotic effect in hepatocytes in contrast to
higher doses. Clearly, further studies are needed to check whether
this dual effect of CA on cell viability can be observed in other cell
types.
Second group of CA effects concerns inflammation. These
effects include the reduction of TNFa via TACE activation,
inhibition of NFkB/p65/p50, suppression of PPARg cleavage,
increase in PGE2 level and inhibition of NO release (Table 2).
Finally, antioxidative properties of CA are thrown into relief by the
increase of GST-P and the reduction of the redox protein p66shc
expression (Table 2). Again, CA at higher doses can induce ROS
generation in some type of cells, while cell pretreatment with
lower doses protects them from the oxidant stress (Tables 1 and 2).
Taken as a whole, this review highlights the need for further
studies to better understand the mechanisms of action of CA in
some forms of human diseases including cancer. It is important to
show first its bioavailability in human body before any human
clinical studies could be performed. Nonetheless, carnosic acid
shows its strong relevance to the treatment of many health
disorders and finally it could find its way to the list of accepted
drugs in phytotherapy.
 S. Bahri et al. / Biomedicine & Pharmacotherapy 84 (2016) 569–582 579

Table 1
Summary of different molecular pathways involved in the anticancer effect of carnosic acid.

Type of CA dose Effect on target cancer cells Molecular pathway involved Ref.
cancer
Colorectal 20– Induction of HCT 116 cells apoptosis ROS generation, inhibition of STAT3 [10]
cancer 100 mM
1– Inhibition of HT-29 cells proliferation Inhibition of AKT and ERK [11]
10 mM
Renal 10– Human renal carcinoma CaKi cells apoptosis ROS generation, TRAIL mediated apoptosis [25]
cancer 20 mM
Brain 10– Human neuroblastoma IMR-32 cells apoptosis Inhibition of ERK and activation of p38 and JNK, [28]
cancer 40 mM ROS generation
Leukemia 2.5– G1 cell cycle arrest/apoptosis on HL-60 and U937 human myeloid leukemia cells in the Activation of cyclin dependant kinase inhibitors [5]
7.5 mM presence of 100 mM of curcuma p21 WAFI and p27 Kipl
15 mM G1 cell cycle arrest/apoptosis on HL-60 human myeloid leukemia cells, in the presence Activation of PTEN/AKT [34]
of 4 mM of As2O
Skin cancer 5– Inhibition of murine B16-F10 melanoma cells adhesion and migration Inhibition of AKT [39]
10 mM
Prostate 20– PC-3 cells apoptosis Modulation of AKT/IKK/NF-kB [40]
cancer 100 mM
Liver cancer 20– HepG2 cells autophagy Inhibition of Akt/mTOR pathway [13]
100 mM
5– HepG2 cells apoptosis Inhibition of Akt/mitochondrial dysfunction [14]
100 mM

Table 2
Signaling pathways modulated by carnosic acid.

Target CA Cell type Physiological effect Ref.

dose
Cell survival Sirtuin-1 activation 10 mM L02 cells CA induced a decrease in the release of cytochrome C, AIF and in [46]
mitochondrial apoptosis induced by ethanol
1 mM Primary hepatocytes and CA reversed the increase in ROS level, LDH release and the decrease in cell [48,49]
HepG2 cells viability produced by H2O2
Keap-1/Nrf2 activation 10 mM COS7 and PC12 h cells CA protects neurons from oxidative stress and glutamate-induced [74]
excitotoxicity
PI3 K/AKT activation 1 mM SH-SY5Y cells CA prevents the effects of paraquat on cell viability and redox parameters [69]
MAPK-p38/JNK activation 1 mM SH-SY5Y cells CA reversed the reduction of Bcl-2/Bax ratio, induction of caspase 3 and [70]
PARP cleavage by 6-OHDA
MAPK-p38/JNK inhibition, 50 mg/ Myocardial cells CA reduced myocardial damage and apoptosis [51]
ERK1/2 activation kg

Inflammation Reduction of TNFa level
TACE activation
NFkB/p65/p50 inhibition
PPARg cleavage suppression
Increase in PGE2 level
Inhibition of NO release
50 mg/ Myocardial cells
kg
30 mM SH-SY5Y cells
20 mM aortic smooth muscle
cells
20 mM U937 cells
10 mM SH-SY5Y cells
10 mM Gastric adeno-carcinoma
cell
20 mM Keratinocyte HaCaT cells
CA reduced myocardial impairment [51]
CA reduced beta-amyloid secretion [57]
CA inhibited vascular smooth muscle migration [52]
CA reduced U937 cells adhesion to HUVECs [53]
CA reduced beta-amyloid oligomer cellular content [58]
CA protected against cell lesion induced by sodium taurocholate [97]
CA protected against skin inflammatory responses [102]

Oxidative Increase of GST-P level 1 mM SH-SY5Y cells CA prevented 6-OHDA-induced apoptosis and oxidative stress [66]
stress
Reduction of p66shc 1 mM HepG2 human hepatoma CA decreased p66shc protein expression induced by ethanol [47]
expression cells

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