Nutrients 08 00731

nutrients
Review
Anticancer Effects of Rosemary
(Rosmarinus officinalis L.) Extract and
Rosemary Extract Polyphenols
Jessy Moore 1 , Michael Yousef 1 and Evangelia Tsiani 1,2, *
1 Department of Health Sciences, Brock University, St. Catharines, ON L2S 3A1, Canada;
jessy.moore@brocku.ca (J.M.); my11dq@brocku.ca (M.Y.)
2 Centre for Bone and Muscle Health, Brock University, St. Catharines, ON L2S 3A1, Canada
* Correspondence: ltsiani@brocku.ca; Tel.: +1-905-688-5550 (ext. 3881)
Received: 23 August 2016; Accepted: 8 November 2016; Published: 17 November 2016
Abstract: Cancer cells display enhanced growth rates and a resistance to apoptosis. The ability
of cancer cells to evade homeostasis and proliferate uncontrollably while avoiding programmed
cell death/apoptosis is acquired through mutations to key signaling molecules, which regulate
pathways involved in cell proliferation and survival. Compounds of plant origin, including food
components, have attracted scientific attention for use as agents for cancer prevention and treatment.
The exploration into natural products offers great opportunity to evaluate new anticancer agents
as well as understand novel and potentially relevant mechanisms of action. Rosemary extract
has been reported to have antioxidant, anti-inflammatory, antidiabetic and anticancer properties.
Rosemary extract contains many polyphenols with carnosic acid and rosmarinic acid found in highest
concentrations. The present review summarizes the existing in vitro and in vivo studies focusing on
the anticancer effects of rosemary extract and the rosemary extract polyphenols carnosic acid and
rosmarinic acid, and their effects on key signaling molecules.
Keywords: rosemary extract; carnosic acid; rosmarinic acid; cancer; proliferation; survival;
cell signaling
1. Introduction
Arguably the most fundamental traits of cancer cells are their enhanced proliferative and
decreased apoptotic capacities [1]. Normal cells tightly control the production and release of
growth factors, which regulate cell growth/proliferation, thereby ensuring cellular homeostasis and
maintenance of normal tissue architecture. In cancer cells, these signals are deregulated and thus,
homeostasis within the cell is disrupted. Proliferation of cancer cells may be enhanced in a number
of ways. Cancer cells may produce growth factors to which they can respond via the expression of
cognate receptors. The level of receptor proteins displayed on the surface of cancer cells can also
be elevated, rendering these cells hyperresponsive to growth factors; the same outcome can result
from alterations to the receptor molecules that facilitate activation of downstream signaling pathways
independent of growth factor binding [1]. Alternatively, cancer cells can signal normal neighbouring
cells resulting in mutations/alterations in signaling pathways. These alterations stimulate the release
of growth factors which are supplied back to the cancer cells, enhancing their proliferation [2,3].
Growth factor receptors (GFR), such as epidermal GFR (EGFR) are plasma membrane proteins with
intrinsic tyrosine kinase (TK) activity. Growth factor binding enhances the tyrosine kinase activity
of the receptor causing receptor autophosphorylation. The phosphorylated tyrosine residues of the
receptor act as docking sites for intracellular proteins containing Src-homology 2 (SH2) domains,
leading to stimulation of intracellular signaling cascades such as the phosphatidylinositol 3-kinase
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Nutrients 2016, 8, 731 2 of 32
(PI3K-Akt) and the Ras-mitogen activated protein kinase (Ras-MAPK) cascades, that result in enhanced
proliferation and inhibition of apoptosis/enhanced survival.
The development of cancer is divided into three stages: initiation, promotion and progression.
Initiation involves a change to the genetic makeup of a cell which primes the cell to become cancerous.
During the stage of promotion various factors permit a single mutated cell to survive (resist apoptosis)
and replicate, promoting growth of a tumor. Finally, as the cancerous cell replicates and develops
into a tumor, the disease state progresses. As normal, healthy cells progress to a neoplastic state they
acquire a series of hallmark capabilities which enable them to become malignant. The 6 hallmarks
of cancer proposed by Hanahan and Weinberg include sustaining proliferative signaling, evading
growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis,
and activating invasion and metastasis [1]. As tumors progress and become more aggressive
they will begin to exhibit more of these hallmarks. Current anticancer agents may be classified
as chemopreventive or chemotherapeutic depending on which stage of carcinogenesis they target.
To explore the chemopreventive potential of anticancer agents, cells in culture or animal models can be
exposed to an anticancer agent before being exposed to a carcinogen. This provides evidence of the
effect of an anticancer agent on the initiation and promotion stages of cancer. Alternatively, cells in
culture or animal models may be exposed to a carcinogen to establish a neoplastic state prior to being
treated with an anticancer agent and this provides evidence of the effect of an anticancer agent on the
progression of cancer.
Many pharmaceutical agents have been discovered by screening natural products from plants.
Some of these drugs such as the chemotherapeutics etoposide, isolated from the mandrake plant and
Queen Anne’s lace, and paclitaxel and docetaxel, isolated from the wood and bark of the Nyssaceae tree,
are currently successfully employed in cancer treatment [4]. The exploration into natural products offers
great opportunity to evaluate new chemical classes of anticancer agents as well as study novel and
potentially relevant mechanisms of action. Many labs, including ours have shown metformin, a drug
derived from the lilac, has anticancer properties [5]. In addition, the polyphenol resveratrol, found
in high concentrations in wine, has been shown to have anticancer effects in vitro and in vivo [6–10].
Importantly, metformin and resveratrol exhibit both chemopreventive and chemotherapeutic effects.
The plant Rosmarinus Officinalis L. a member of the mint family Lamiaceae, is native to the
Mediterranean region and has many culinary and medicinal uses. The main polyphenols found
in rosemary extract (RE) include the diterpenes carnosic acid (CA) and rosmarinic acid (RA) [11].
Rosemary extract and its polyphenols CA and RA have recently been explored and found to exert
potent anticancer effects (reviewed recently in [12–14]). To establish a systematic literature review we
used the online search engine Pubmed. We searched the key phrases: rosemary extract and cancer,
carnosic acid and cancer, rosmarinic acid and cancer. We also included subtypes of cancer such as breast
cancer, colon cancer, etc., as keywords in our search. All studies pertaining to our topic and published
after the year 2000 were included in the current review. In the following sections, in vitro and in vivo
studies on the effects of RE and its main polyphenols have been summarized and sorted by cancer
cell type, in chronological order from earliest to most recent. Chronology was chosen as the sorting
method to highlight how the literature has progressed and what knowledge is currently available.
Initially we focused on the studies examining the anticancer effects of RE, we then highlighted studies
in which mechanisms of action have been investigated and separately summarized the studies using
the polyphenols CA and RA. The studies presented in the text are also summarized, organized and
presented in a table format to allow the reader to extract the information easily.
This is a comprehensive systematic review and adds to the existing literature by summarizing all
relevant studies using RE, CA and RA in each cancer subtype. The review is organized by experimental
treatment (RE, CA, RA), type of cancer (histology) and the study model (in vitro or in vivo) resulting
in a clear, detailed and inclusive summary of the existing literature. This review also focuses on the
mechanistic data provided by these studies, which will be beneficial for future research to help focus
efforts on identifying the main mechanisms involved in the anticancer action of RE, CA and RA.
Nutrients 2016, 8, 731 3 of 32
2. Anticancer Effects of Rosemary Extract (RE): In Vitro Studies

Several in vitro studies using colon cancer cell lines have shown RE to exhibit anticancer properties
(Table 1). Exposure of CaCo-2 colon cancer cells to RE drastically decreased colony formation at
30 µg/mL (24 h) [15]. Yi, et al. (2011) examined the anti-tumorigenic effect of several culinary and
medicinal herbs on SW480 colon cancer cells and found RE to significantly decrease cell growth at a
concentration of 31.25 µg/mL (48 h), with an IC50 of approximately 71.8 µg/mL [16]. Cell proliferation
was dramatically decreased and cell cycle arrest was induced in HT-29 and SW480 cells using extracts
that were standardized to CA (25%–43%) or to total polyphenol content (10 µM) [17–19]. Cell growth
of SW620 and DLD-1 colon cancer cells was significantly inhibited by RE at 30 µg/mL (48 h), with an
IC50 as low as 34.6 µg/mL. Furthermore, RE enhanced the inhibitory effects of the chemotherapeutic
drug 5-fluorouracil (5-FU) on proliferation and sensitized 5-FU resistant cells [20].
In SW620 and DLD-1 colon cancer cells RE inhibited cell viability dose-dependently resulting
in significant inhibition at concentrations as low as 20 µg/mL, and an IC50 around 25 µg/mL (48 h).
This study used 5 different RE’s, containing increasing levels of carnosol (CN: 1%–3.8% w/w) and CA
(10%–30% w/w). Inhibition of cell viability was correlated with increasing CA content. Furthermore,
CA alone (at doses found in RE) decreased cell viability and this effect was potentiated by the
addition of CN (at doses found in RE). However, the inhibition seen using RE was greater than the
response seen with CA or CN alone or in combination suggesting that chemicals other than CA and
CN present in RE, also contribute to its anticancer effects [21]. Similarly, RE inhibited cell viability
in HT29, SW480 and HGUE-C-1 colon cells at comparable doses (1.5–100 µg/mL; 48 h) and the
authors reported that individual fractions of RE containing CA and other polyphenols, while potent,
were not as potent as the complete extract [22]. Using HCT116 and SW480 cells, 10–100 µg/mL RE
standardized to 23% CA (24–72 h) inhibited cell viability and induced apoptosis [23]. Valdes, et al.
have shown, using HT-29 colon cells, that 30–60 µg/mL RE (24–72 h) inhibits cell proliferation
(IC50 16.2 µg/mL). Moreover, RE induced cell cycle arrest, necrosis, cholesterol accumulation and ROS
accumulation [24–26]. These studies provide evidence for the role of RE as an anticancer agent in colon
cancer cells, capable of consistently inhibiting cell growth and viability at relatively low concentrations
in the 20–100 µg/mL range.
Table 1. Anticancer effects of Rosemary Extract (RE). In vitro studies: colon cancer.
Cancer Cell Dose/Duration Findings Mechanism Reference

↓ H2 O2 -induced DNA strand
↓ cell colony formation.
CaCo-2 (Colorectal breaks and oxidative damage.
0.1–30 µg/mL (3–24 h) Long and short term [15]
adenocarcinoma) ↓ visible light-induced
antioxidant effects
oxidative damage
↓ cell proliferation.
SW480 (Colorectal Cytotoxic above
31.25–500 µg/mL (48 h) [16]
adenocarcinoma) 250 µg/mL.
IC50~71.8 µg/mL
↓ cell proliferation
HT-29 (Colorectal RE containing 10 µM
↑ cell cycle arrest [17]
adenocarcinoma) total polyphenols (72 h)
↑ apoptosis
1.95–62.5 µg/mL (48 h)
HT29 (Colorectal ↓ cell proliferation
3 RE’s standardized to [18]
adenocarcinoma) IC50 > 62.5 µg/mL
25.9%, 36.2%, 42.4% CA
SW480 (Colorectal ↑ antioxidant and xenobiotic
adenocarcinoma), RE containing 10 µM effects Modulates: Nrf2, ER
SW480 more sensitive [19]
HT29 (Colorectal total polyphenols (48 h) stress genes, cell cycle,
↑ cell cycle arrest
adenocarcinoma) proliferation genes
SW620 (Colorectal IC50 36.4 and 34.6 µg/mL
adenocarcinoma), Effect on 5-FU sensitive Modulates TYMS and TK1.
20–110 µg/mL (24–48 h) [20]
DLD-1 (Colorectal and resistant cells ↑ PARP cleavage
adenocarcinoma) ↑ apoptosis
↓ cell transformation
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Table 1. Cont.

SW620 (Colorectal
↑ PARP cleavage.
adenocarcinoma), ↓ cell viability
20–120 µg/mL (48 h) ↑ GCNT3. [21]
DLD-1 (Colorectal IC50 25 µg/mL
↓ miR-15b gene expression
adenocarcinoma)
HT-29 (Colorectal
adenocarcinoma),
W480 (Colorectal
1.5–100 µg/mL (24–48 h) ↓ cell viability [22]
adenocarcinoma),
HGUE-C-1 (Colorectal
carcinoma)
↑ Nrf2
HCT116 (Colorectal
10–100 µg/mL ↑ PERK
carcinoma), SW480 ↓ cell viability
(24 h, 48 h, 72 h) ↑ sestrin-2 [23]
(Colorectal ↑ apoptosis
Standardized to 23% CA ↑ HO-1
adenocarcinoma)
↑ cleaved-casp 3
↑ UPR
↑ ER-stress
HT-29 (Colorectal ↑ cell cycle arrest
30 µg/mL (2–72 h) ↓ cell cycle genes [24]
adenocarcinoma) ↑ cholesterol accumulation
Altered
↑ ROS accumulation
cholesterol-modulating genes
↓ cell proliferation ↑ Nrf2 pathway
HT-29 (Colorectal
30–70 µg/mL (24 h, 48 h) IC50 16.2 µg/mL ↑ UPR [25]
adenocarcinoma)
↑ necrosis ↑ autophagy
↑ H2 O2 in media
HT-29 (Colorectal ↓ cell proliferation
30–60 µg/mL (6 h, 24 h) ↑ ROS levels [26]
adenocarcinoma) ↑ cell cycle arrest
↑ HO-1 and CHOP expression
H2 O2 (hydrogen peroxide), 5-FU (fluorouracil), TYMS (thymidylate synthase), TK1 (thymidine kinase 1), PARP
(poly ADP ribose polymerase), GCNT3 (glucosaminyl (N-acetyl) transferase 3), miR-15b (microRNA-15b). GI50
(50% growth inhibition), TGI (total growth inhibition), Nrf2 (nuclear factor erythroid 2-related factor 2), casp
(caspase), UPR (unfolded protein response), ER (endoplasmic reticulum), HO-1 (heme oxygenase protein-1),
CHOP (C/EBP homologous protein).
In rat RINm5F insulinoma cells, RE significantly inhibited cell proliferation at 25 µg/mL (24 h),
viability at 12 µg/mL (24 h) and increased apoptosis at 25 µg/mL (24 h) [27] (Table 2). Exposure
of pancreatic cancer cells PANC-1 and MIA-PaCa-2 to RE containing increasing concentrations of
CN (1%–3.8% w/w) and CA (10%–30% w/w) resulted in significant inhibition of cell viability with
an IC50 of 50 µg/mL (48 h) and 30 µg/mL (48 h) respectively. The RE containing 25.66% w/w CA
(sub-max) caused maximal inhibition compared to other RE’s in PANC-1 cells, significantly inhibiting
cell viability to approximately 60% at 40 µg/mL (48 h) [21].
Breast cancer can be classified under three subtypes based on the sensitivity of the tumors to
chemotherapeutic agents. The subtypes are (i) estrogen receptor positive (ER+), which express ERα
and therefore respond to estrogens; (ii) human epidermal growth factor receptor 2 positive (HER2+)
which overexpress HER2 and can be either ER+ or ER−; (iii) triple negative (TN) which lack expression
of ERα, progesterone receptor and HER2. One study used MCF-7 (ER+) breast cancer cells and a
cigarette smoke solution (in PBS) collected from a cigarette with or without 40 mg RE added to the
filter. The control used in this experiment was cells stimulated with 2.5 µM benzopyrene for 12–18 h
and exposed to 1:19 v/v cigarette smoke solution for 2 h without an RE filter. The presence of RE in
the filter lead to considerably reduced benzopyrene levels and associated DNA adduct formation [28]
(Table 2).
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RE inhibited cell proliferation in breast cancer cells with an IC50 of 90 µg/mL and 26.8 µg/mL
in MCF-7 (ER+) and MDA-MB-468 (TN) cell lines respectively [29] (Table 2). In a similar study,
dose-dependent inhibition of cell viability by 6.25–50 µg/mL (48 h) RE was seen in MDA-MB-231
(TN) and MCF-7 (ER+) breast cancer cells and MCF-7 cells had an IC50 of ~24.02 µg/mL. There is
a discrepancy seen in the reported IC50 values which may be attributed to the different extraction
methods used for the preparation of rosemary extract; supercritical CO2 [30] and ethanol extraction [29].
Furthermore, MCF-7 cells were used in 2 additional studies and while both were found to inhibit
cell proliferation, the IC50 values varied greatly from 187 µg/mL [31] to 9.95–13.89 µg/mL
(RE standardized to 25%–43% CA) [18]. In agreement with the aforementioned studies, the RE
resulting in a higher IC50 value was obtained from an alcohol based, methanol extraction [31].
The effects of RE at 1–120 µg/mL (48 h) were explored in all three breast cancer subtypes, ER+,
HER2+ and TN. RE caused dose-dependent inhibition of cell viability in all subtypes of breast cancer
cells. Furthermore RE enhanced the effectiveness of the monoclonal antibody (mAb) trastusumab
and the chemotherapeutic drugs tamoxifen and paclitaxel, used in the treatment of breast cancer [32].
Taken together, these studies suggest a role for RE to inhibit pancreatic and breast cancer cell viability
and proliferation, and induce apoptosis at concentrations in the 10–100 µg/mL range.
Table 2. Anticancer effects of Rosemary Extract (RE). In vitro studies: pancreatic and breast cancer.

↑ nitrate accumulation.
RINm5F (Insulinoma) 12–100 µg/mL (24–48 h) ↓ cell viability [27]
↑ TNFα production.
↑ apoptosis
MIA-PaCa-2 (Pancreatic
carcinoma), PANC-1 20–120 µg/mL (48 h) ↓ cell viability ↑ PARP-cleavage [21]
(Pancreatic carcinoma)
40 mg RE powder filter
MCF-7 (ER+) ↓ BP levels and associated
(inserted into cigarette) [28]
(Breast adenocarcinoma) DNA adduct formation.
(2 h)
MCF-7 (ER+)
(Breast adenocarcinoma), IC50 ~90 µg/mL and
0.1%–20% (5–120 h) [29]
MDA-MB-468 26.8 µg/mL
(Breast adenocarcinoma)
MCF-7 (ER+)
(Breast adenocarcinoma), ↓ cell viability IC50
6.25–50 µg/mL (48 h) [30]
MDA-MB-231 ~20.42 µg/mL
MCF-7 ↓ cell proliferation IC50
1–250 µg/mL (48 h) [31]
(Breast adenocarcinoma) 187 µg/mL
1.95–62.5 µg/mL (48 h)
MCF-7 ↓ cell proliferation IC50
3 REs standardized to [18]
(Breast adenocarcinoma) 9.95–13.89 µg/mL
25.9%, 36.2%, 42.4% CA
SK-BR-3 (HER2+)
(Breast adenocarcinoma),
UACC-812 (HER2+) ↑ FOS levels
↓ cell viability Enhanced
(Breast ductal carcinoma), ↑ PARP cleavage
effect of chemotherapeutics
T-47D (ER+) (Breast ductal 10–120 µg/mL (48 h) ↓ HER2 [32]
↑ apoptosis
carcinoma), MCF-7 (ER+) ↓ ERBB2
↓ cell transformation
(Breast adenocarcinoma), ↓ ERα receptor.
MDA-MB-231
TNFα (tumor necrosis factor), PARP (poly ADP ribose polymerase), BP (benzopyrene), Fos (FBJ murine
osteogenic sarcoma virus), HER2 (human epidermal growth factor receptor 2), ERBB2 (HER2/neu gene), ERα
(estrogen receptor α).
Rosemary extract (6.25–50 µg/mL; 48 h) inhibited viability of DU145 and PC3 prostate cancer
cells [30] (Table 3). In agreement with these data, significant inhibition of LNCaP and 22RV1
prostate cancer cell proliferation and viability, and an induction of apoptosis were seen with RE
(50 µg/mL standardized to 40% CA; 24–48 h) [33]. RE was able to combat the enhanced prostate
Nutrients 2016, 8, 731 6 of 32
specific antigen (PSA) levels measured in cell culture media, indicative of prostate cancer, inhibiting
levels to less than a fifth of what was seen in the control group. Correspondingly, levels of the androgen
receptor, to which PSA binds, were significantly decreased by 50 µg/mL RE [33]. The inhibitory
effects on both androgen sensitive and insensitive cell lines are important and suggest potential
chemotherapeutic effects in different prostate cancer subtypes.
Using 5637 bladder cancer cells Mothana, et al. (2011) showed that RE inhibited cell proliferation
with an IC50 of 48.3 µg/mL (48 h) [31] (Table 3). Exposure of A2780 ovarian cancer cells to 0.08%
(0.8 mg/mL; 48 h) RE containing media resulted in significant inhibition of proliferation and induction
of apoptosis and cell cycle arrest. Cisplatin is a chemotherapeutic agent used often in cancer treatment
however, as with many chemotherapeutics, patients often develop resistance to treatment. At 0.08%
RE enhanced the sensitivity of A2780 and cisplatin-resistant A2780CP70 cell lines to growth inhibition
by cisplatin treatment, suggesting that RE may be of use in combination with cisplatin or potentially
other chemotherapeutic drugs in patients who have developed an acquired resistance [34]. In HeLa
cervical cancer cells, RE inhibited cell proliferation with an IC50 of 23.31 µg/mL (72 h) [35] and RE
standardized to CA (25%–43%) inhibited cell proliferation with an IC50 of ~10 µg/mL (48 h) [18],
suggesting that standardized extracts containing higher concentrations of CA may have greater
anticancer effects. Furthermore, in human ovarian cancer cells SK-OV3 and HO-8910 rosemary
essential oil (0.0625%–1%) inhibited cell viability with an IC50 of 0.025% and 0.076% in each cell
line respectively (48 h) (Table 3) [36]. This study noted that the rosemary essential oil was more
potent than its individual components (α-pinene, β-pinene, 1,8-cineole) when tested alone at the
same concentrations.
Table 3. Anticancer effects of Rosemary Extract (RE). In vitro studies: prostate, ovarian, cervical and
bladder cancer.

DU145 (Prostate
↓ cell viability IC50
adenocarcinoma), PC3 6.25–50 µg/mL (48 h) [30]
~8.82 µg/mL
(Prostate adenocarcinoma)
↑ CHOP
↓ PSA production
LNCaP (Prostate 10–50 µg/mL (24–48 h) ↑ cell cycle arrest
↑ Bax
adenocarcinoma), 22RV1 RE standardized to ↑ apoptosis [33]
↑ cleaved-casp 3
(Prostate carcinoma) 40% CA modulates endoplasmic
↓ androgen receptor
reticulum stress proteins.
expression
↓ cell proliferation IC50
5637 (Bladder carcinoma) 0–250 µg/mL (48 h) [31]
48.3 µg/mL
↓ cell proliferation Enhanced
sensitivity of cisplatin
A2780 (Ovarian carcinoma),
-resistant cell lines. ↓ P-glyco protein
A2780CP70
0.05%–0.25% (24–48 h) ↑apoptosis ↑ cytochrome c gene [34]
(cisplatin-resistant)
↑ cell cycle arrest ↑ hsp70 gene
(Ovarian carcinoma)
Modulates expression of
apoptotic genes.
HeLa (Cervical ↓ cell proliferation IC50
1.56–400 µg/mL (72 h) [35]
adenocarcinoma) 23.31µg/mL
1.95–62.5 µg/mL (48 h)
HeLa (Cervical ↓ cell proliferation IC50
3 REs standardized to [18]
adenocarcinoma) 10.02–11.32 µg/mL
25.9%, 36.2%, 42.4% CA
SK-OV3 (Ovarian ↓ cell viability
0.0625%–1% rosemary
adenocarcinoma), HO-8910 IC50 0.025% (SK-OV3) [36]
essential oil (48 h)
(Ovarian carcinoma) IC50 0.076% (HO-8910)
CHOP (C/EBP homologous protein), PSA (prostate specific antigen), Bax (Bcl-2 associated X protein), casp
(caspase), hsp70 (heat shock protein 70).
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In human liver Hep-3B cells, RE at 0–50 µg/mL (24–48 h) dose-dependently decreased cell
viability [30,37] with an IC50 of 22.88 µg/mL [30] (Table 4). Cell viability was inhibited in Bel-7402
liver cells by rosemary essential oil with an IC50 of 0.13% (1.3 mg/mL; 48 h) [36] and in HepG2
liver cells by RE with an IC50 of 42 µg/mL (48 h) [38]. The latter study also found that of the
4 different extracts tested, those with higher concentrations of CA resulted in more potent inhibition of
cell proliferation [38]. In lung cancer cells, RE decreased viability of NCI-H82 small cell carcinoma
cells (6.25–50 µg/mL; 48 h) [30] and decreased proliferation of A549 non-small cell carcinoma cells
(2.5–200 µg/mL) [39] with an IC50 or 24.08 µg/mL and 15.9 µg/mL in each cell line respectively
(Table 4). In a V79 normal hamster lung fibroblast cell line RE was cytotoxic at 30 µg/mL (24 h) [15].
The cytotoxicity of RE in normal fibroblasts raises questions about its potential as a successful treatment
option however, further research is required to fully examine the cytotoxicity issue in normal tissues.
Table 4. Anticancer effects of Rosemary Extract (RE). In vitro studies: liver and lung cancer.

Hep-3B
0.5–5 µg/mL (24 h) ↓ cell viability ↑ TNFα [37]
(Hepatocellular carcinoma)
Hep-3B ↓ cell viability
6.25–50 µg/mL (48 h) [30]
(Hepatocellular carcinoma) IC50 ~22.88 µg/mL
Bel-7402 0.0625%–1% rosemary ↓ cell viability
[36]
(Hepatocellular carcinoma) essential oil (48 h) IC50 0.13%
↓ cell viability
HepG2
10–120 µg/mL (48 h) IC50 42 µg/mL [38]
GI50 20 µg/mL
NCI-H82 ↓ cell viability
6.25–50 µg/mL (48 h) [30]
(Lung carcinoma; SCLC) IC50 ~24.08
↓ H2 O2 -induced DNA
Cytotoxic to cells at
strand breaks and
30 µg/mL (24 h) Long
V79 (Normal hamster lung) 0.1–30 µg/mL (3–24 h) oxidative damage. [15]
and short term
↓ visible-light induced
antioxidant effects
oxidative damage
↓ p-Akt
A549 ↓ p-mTOR
2.5–200 µg/mL (48–72 h) ↓ cell survival [39]
(Lung adenocarcinoma) ↓ p-P70S6K
↑ apoptosis IC50 ~15.9
↑ PARP cleavage
mTOR (mammalian target of rapamycin), PARP (poly(ADP-ribose) polymerase).
Vitamin D analogues (VDA) are commonly used in clinical differentiation therapy of acute
myeloid leukemia (AML) to attempt to restore a defect in the capacity of myeloid progenitor cells
to mature into non-replicating adult cells. However, pharmacologically relevant doses have been
found to result in many adverse events such as hypercalcemia and attempts to circumvent these
adverse events have been unsuccessful. RE containing 10 µM equivalent of CA, or 10 µM CA alone
(96 h) potentiated the ability of vitamin D derivatives to inhibit cell viability and proliferation, induce
apoptosis and cell cycle arrest and increase differentiation of WEHI-3BD murine leukemic and human
HL-60 leukemic cells [40,41] (Table 5). A study examining the human leukemia HL-60 and K-562
cell lines and the murine RAW264.7 macrophage/monocyte cell line found significant inhibition of
proliferation with an IC50 of 0.14% (1.4 mg/mL) and 0.25% (2.5 mg/mL) for the HL-60 and K-562
cells, respectively. In addition 0.1% (1 mg/mL; 72 h) RE significantly increased differentiation of HL-60
cells [29]. RE inhibited viability at 50 µg/mL (48 h) in K-562 leukemia cells [30]. Similar effects of RE
(50 µg/mL; 24 h) were reported by others that lead to decreased proliferation of K-562 cells [42].
Nutrients 2016, 8, 731 8 of 32
Table 5. Anticancer effects of Rosemary Extract (RE). In vitro studies: leukemia.

Potentiated following effects
WEHI-3B D (Murine of VDA:
myeloid leukemia), HL-60 RE (10 µM equivalent of ↓ cell proliferation
↑ G1 phase [41]
(Myeloid leukemia), U937 CA) (48–96 h) ↑ cell cycle arrest
(Myeloid leukemia) ↑ cell differentiation
↑ apoptosis
↓ cell proliferation IC50
↓ (LS) NO
RAW 264.7 (Murine ~18.76 µg/mL and
↑ antioxid-ant activity
leukemia; macrophage), 0.1%–20% (5–120 h) 33.5 µg/mL
↔ basal TNFα, IL-1β, [29]
HL-60 (Myeloid leukemia), (1–200 mg/mL) ↑ cell differentiation
iNOS or COX2
K-562 (Human leukemia) ↓ LPS-stimulated (LS)
↓ (LS) IL-1β and COX2
antioxidant activity
Potentiated following effects
↓ ROS
of VDA:
WEHI-3B D (Murine RE (10 µM equivalent of ↑ antioxid-ant activity
↑ cell differentiation [40]
myeloid leukemia) CA) (48–96 h) ↑ NADP(H)-quinone
↓ cell viability
reductase
↓ cell viability IC50
K-562 (Human leukemia) 6.25–50 µg/mL (48 h) [30]
~12.50 µg/mL
↓ AKT1
K-562 (Human leukemia),
50 µg/mL (0–96 h) ↓ cell proliferation ↑ Rb2 [42]
U937 (Myeloid leukemia)
↔ ERK2
VDA (vitamin D analogue), LPS (lipopolysaccharide), NO (nitric oxide), TNFα (tumor necrosis factor α),
IL-1β (interleukin 1β), iNOS (inducible nitric oxide synthase), COX2 (cyclooxygenase 2), ROS (reactive oxygen
species), NADP (nicotinamide adenine dinucleotide phosphate), Rb2 (retinoblastoma-related gene 2).
3. Anticancer Effects of Rosemary Extract (RE): In Vivo Animal Studies

A limited number of studies have examined the effects of RE administration on tumor growth
in animals in vivo (Table 6). Administration of RE (1 mg/mL) in the drinking water ad libitum for
32–35 days resulted in a significant decrease in tumor size in nude mice xenografted with SW620
colon cancer cells [21]. A similar study using HCT116 colon cancer xenografted athymic nude mice
fed 100 mg/kg/day RE dissolved in olive oil (4 weeks) significantly decreased tumor size in treated
animals compared to control [23]. Biochemical analysis of serum samples collected from Sprague
Dawley rats with N-methylnitrosourea-induced colon cancer showed significant anticancer effects by
both high (3333.3 mg/kg/day) and low (1666.6 mg/kg/day) dose RE after 4 months of treatment with
significant alteration of gene and protein signaling and aggregation of lymphoid cells [43]. A significant
reduction in tumor volume was seen in mice xenografted with 22RV1 prostate cancer cells by RE
(100 mg/kg/day) which was administered, dissolved in olive oil for 22 days [33].
In a diethylnitrosamine (DEN)-induced liver cancer model in F344 rats, RE at 100 mg/kg/day
(5 days) was administered intragastrically with an intraperitoneal (i.p) injection of DEN on day 4.
From this point, rats were fed a normal diet for 3 weeks until undergoing partial hepatectomy.
Examination of liver tissue suggested RE may exert some protective antioxidant effects [44].
In accordance with this, use of Swiss mice exposed to 6 Grays (Gy) ionizing radiation (IR) in
their liver once, followed by treatment with 1000 mg/kg RE fed orally, daily for 5 days suggested
protective, antioxidant activity by RE. A delayed onset of IR-induced mortality and attenuated
increases in glycogen and protein levels were seen in livers of mice exposed to IR and fed RE,
compared to IR-exposed mice not fed RE [45]. Caution should be taken however, due to the high
concentration (1000 mg/kg) used [45] which is at least 10 times greater than what has been found to
exert potent anticancer effects in other studies. Taken together, these studies suggest a role for RE
inhibiting chemical- or IR-induced carcinogenesis by exerting protective, antioxidant effects on healthy
tissues. Thus, RE may display radio-protective effects, which would benefit healthy tissue during
radiation treatment.
In WEHI-3BD myeloid leukemia xenografted mice fed 1% w/w RE in their food ad libitum
(29 days), investigators noted a significant decrease in both tumor volume and incidence. Furthermore,
Nutrients 2016, 8, 731 9 of 32
RE showed an additive effect when combined with Vitamin D analogues (VDA) [41]. In WEHI-3BD
xenografted mice administered RE (4% w/w in food) for up to 15 weeks combined with VDAs, median
survival time was significantly increased and white blood cell count decreased to levels comparable to
those seen in the control group of healthy mice [40].
Using a 7,12-dimethylbenz(a)anthracene (DMBA)-induced skin cancer nude mouse model, RE
(500 or 1000 mg/kg/day; 15 weeks) administered orally in water resulted in a significant decrease
in tumor number, diameter, weight and decrease in tumor incidence and burden, and an increase in
latency period compared to control mice treated with DMBA only [46,47]. One group of mice, which
were administered RE for 7 days prior to the first application of DMBA, showed a 50% reduction
in tumor growth compared to the DMBA-only treated mice, suggesting potent chemo protective
effects [47].
Table 6. Anticancer effects of Rosemary Extract (RE). In vivo studies.
Animal Model Dose/Duration Findings Mechanism Reference

1 mg/mL in drinking
SW620 colon xenograft
water (32–35 days) ad ↓ tumor size ↓ miR-15b in plasma [21]
(nude mice)
libitum
100 mg/kg/day in
HCT116 colon xenograft ↑ Nrf2 expression
100 µL olive oil by oral ↓ tumor size [23]
(athymic nude mice) ↑ sestrin-2 expression
gavage (4 weeks)
↑ cyt C
1666.6 mg/kg/day
↑ PCDP4
(low dose) RE or Both RE showed comparable
NMN-induced colon cancer ↓CEA
3333.3 mg/kg/day effects. Lead to lymphoid cell [43]
(Sprague-Dawley rats) ↓ CCSA-4
(high dose) RE orally aggregation in submucosa
↓ β-catenin, K-ras, c-myc
(4 months)
gene expression
↓ androgen receptor
22RV1 prostate xenograft 100 mg/kg/day in olive ↓ tumor volume expression
[33]
(athymic nude mice) oil, orally (22 days) (induces apoptosis) ↓ PSA
↑ CHOP
100 mg/kg/day RE
intragastrically (5 days)
Injected i.p with 20
DEN-induced liver cancer
mg/kg DEN on day 4. ↑ antioxidant activity ↓ GST positive foci [44]
(F344 rats)
Fed normal diet until
week 3 (underwent
partial hepatectomy)
Delayed onset of
6Gy γ-IR (once) followed IR-induced mortality
Swiss mice exposed to ↓ LPx levels
by 1000 mg/kg/day RE Attenuated negative IR effects [45]
γ-IR (liver) ↑ GSH levels
orally (5 days) Protective effect on liver
and blood
↓ tumor volume
Myeloid leukemia 1% RE w/w in food ad ↓ tumor incidence Potentiated
[41]
inoculated mice libitum (29 days) VDA ability to
↓ tumor volume
RE alone ↔ median survival
Myeloid leukemia 4% w/w in food ad
time RE+VDA ↓ WBC [40]
inoculated mice libitum (15 weeks)
↑ median survival time
↓ tumor number
1000 mg/kg/day RE ↓ tumor incidence
DMBA-induced skin cancer ↓ LPx levels
orally in water or by ↓ tumor burden [46]
(nude mice) ↑ GSH levels
gavage (15 weeks) ↓ tumor yield
↑ latency period
500 mg/kg/day RE ↓ tumor number
DMBA-induced skin cancer ↓ LPx levels
orally in water or by ↓ tumor diameter [47]
(nude mice) ↑ GSH levels
gavage (15 weeks) ↓ tumor weight
miR-15b (microRNA 15b), PSA (prostate specific antigen), CHOP (C/EBP homologous protein), VDA
(vitamin D analogue), WBC (white blood cell), GST (glutathione S transferase), IR (ionizing radiation), LPx
(lipid peroxidase), GSH (glutathione), DEN (diethylnitrosamine), DMBA (7,12-dimethylbenz(a)anthracene),
NMN (N-methylnitrosourea), cyt C (cytochrome C), PCDP4 (programmed cell death protein 4), CEA
(carcinoembryonic antigen), CCSA-4 (colon cancer specific antigen 4), LPx (lipid peroxidase), GSH (glutathione).
Nutrients 2016, 8, 731 10 of 32
4. Mechanisms of Anticancer Effects of Rosemary Extract (RE): In Vitro Studies

Many studies have examined the anti-proliferative and colony forming abilities of RE in vitro
in colon [15–20,24–26], pancreas [27], breast [18,29,31,32], prostate [33], cervical [18,35], bladder [31],
ovarian [34], lung [39] and leukemia [29,40–42] cell lines however, little is known concerning the
underlying mechanism. RE was shown to have an inhibitory effect on AKT1 mRNA and protein
expression, a protein involved in the PI3K/Akt survival signaling pathway, in a leukemic cell
line [42] however, no measure of Akt activity was mentioned. No effect on ERK2 protein levels,
involved in cell proliferation and differentiation, were seen in these cells. Cell cycle arrest prevents
further division by proliferating cells and RE was shown to induce cell cycle arrest in a number of
cancer cell lines [17,19,24,25,33,34,41] and increase retinoblastoma-related gene 2 (Rb2) [42] which
regulates entry into cell division. Recently, Moore, et al. (2016) found RE inhibited activation of
the Akt/mTOR/p70S6K signaling pathway which was associated with a significant decrease in cell
proliferation and survival [39].
The viability of various cancer cell lines was shown to be significantly inhibited by treatment with
RE which many studies attributed to enhanced apoptosis and cell death. Increased poly ADP ribose
polymerase (PARP) cleavage, which is an established indicator of enhanced apoptosis, was seen in
colon [20,21], pancreas [21], breast [32] and lung [39] cancer cell lines following treatments with RE.
Alternatively, RE enhanced nitrate accumulation (i.e., increased nitric oxide production) and TNFα
production in pancreatic [27] and liver [37] cancer cells, indicative of enhanced cell death capabilities
and nitric oxide-induced apoptosis. In ovarian cancer cells [34] enhanced apoptosis was associated with
increased gene expression of mitochondrial-regulated apoptosis proteins cytochrome c, involved in the
electron transport chain, and heat shock protein 70 (hsp70) which is involved in protein folding and
protecting the cell from heat stress and toxic chemicals. Other mechanisms of apoptosis by RE include
enhanced protein expression of pro-apoptotic Bax and cleaved-caspase 3 [23,33], increased expression
of binding immunoglobulin protein (BiP) and CCAAT/-enhancer-binding protein homologous
protein (CHOP) proteins which induce endoplasmic reticular stress [25,33], and the unfolded protein
response [24–26,33] in prostate and colon cancer cells. Interestingly, in normal prostate epithelial
cells RE treatment resulted in a decrease in endoplasmic reticular stress related protein PRKR-like
endoplasmic reticulum kinase (PERK), suggesting RE selectively induces endoplasmic reticular stress
in prostate cancer cells but spares normal prostate cells [33]. Similarly, in breast cancer cells [32] RE
decreased expression of estrogen receptor α (ERα) in the ER+ subtype and human epidermal growth
factor receptor 2 (HER2) in the HER2+ subtype, and it was suggested the decreased receptor expression
was correlated with enhanced apoptosis in these cell subtypes. Correspondingly, increased levels of
Fos, an oncogenic transcription factor, were detected in ER+ and HER2+ cell lines, and this event is
thought to precede apoptosis and correspond to the PARP-cleavage seen in these cells. Although RE
was also capable of inducing anticancer effects in triple negative (TN) breast cancer cells, its mechanism
has yet to be elucidated [32].
Induction of apoptosis by endoplasmic reticular stress has been found by several studies
in colon cancer cells [19,23,24,26] and has been shown to involve translocation of nuclear factor
erythroid 2-related factor 2 (Nrf2) into the nucleus and induction of p38 MAPK and PERK activity.
The Nrf2/antioxidant response element (ARE) signaling pathway has been considered to protect cells
against carcinogenesis and attenuate cancer development by neutralizing ROS and carcinogens and
members of this pathway, including sestrin-2 and heme oxygenase-1 (HO-1), are upregulated by RE in
colon cancer cells [23,25]. Overall, the majority of existing studies indicate that the anticancer effects of
RE may be due largely to induction of apoptosis.
Antioxidants are molecules, which scavenge harmful free radicals, protecting cells from oxidative
DNA damage and potentially death. RE has been shown to exert antioxidant effects in colon [15],
breast [28], and leukemia [29,40] cell lines. Colon cancer cells pretreated with RE followed by
treatment with hydrogen peroxide, often used in cell culture to induce oxidative DNA damage,
showed reduced DNA double-strand breaks and oxidative damage compared to control cells treated
Nutrients 2016, 8, 731 11 of 32
with hydrogen peroxide only. Similarly, RE reduced oxidative damage induced by methylene blue
(oxidizes purines) in these cells [15]. RE treatment resulted in increased levels of antioxidants and
NAPD(H)-quinone reductase (oxidoreductase involved in the transfer of electrons from a reduced
molecule to an oxidized molecule) which decreased reactive oxygen species (ROS) levels, and inhibited
lipopolysaccharide (LPS)-stimulated production of the free radical nitric oxide (NO) in leukemia cell
lines [29,40]. In an in vitro model of cigarette smoking, the use of an RE containing cigarette filter
considerably reduced benzopyrene (carcinogen) levels and associated DNA adduct formation in breast
cancer cells [28]. An effect of RE treatment, to inhibit ROS levels in cancer cells, may be viewed as a
beneficial and not an anticancer effect for cancer cells. Traditionally treatments for cancer should result
in apoptosis/killing of cancer cells. The antioxidant properties exerted by RE treatment indicate a
potential for RE as a preventative strategy which may target the initiation and promotion stages of
cancer. Antioxidants work to restore damaged DNA back to normal and protect the cell from further
damage thus, preventing the potential mutation into a cancer cell and subsequent tumor formation.
In addition to the antiproliferative, apoptotic and antioxidant mechanisms noted above, some
evidence indicates that RE may (i) exert anti-inflammatory effects [29] through inhibition of
interleukin-1 (IL-1) and cyclooxygenase 2 (COX2) molecules; (ii) aid in the reversal of acquired
drug resistance [34] by inhibiting P-glycoprotein levels (involved in drug resistance); and (iii) alter
metabolic-related genes [21] such as glycosyltransferase (GCNT3) which forms glycosidic linkages
in a variety of macromolecules and its potential epigenetic regulator microRNA-15b. Induction of
autophagy [26] and alterations to cholesterol metabolism [24] may also be mechanisms of RE in colon
cancer cells.
5. Mechanisms of Anticancer Effects of Rosemary Extract (RE): In Vivo Animal Studies

Limited evidence exists regarding RE’s mechanism in vivo however, few studies list potential
antioxidant effects and serum biomarkers for RE’s anticancer effects. Increases in glutathione (GSH),
an antioxidant, and reductions in lipid peroxidase (LPx), an oxidizing agent resulting in free radical
production and cell damage, have been recorded in IR-induced mouse liver [45] and DMBA-induced
mouse skin cancer [46,47] models treated with RE. Similarly, RE decreased glutathione-S transferase
(GST) positive foci, which are associated with oxidative damage from the reduction of GSH [40],
in a rat DEN-induced liver cancer model however, results were not significant and should be taken
with caution [44].
Serum samples from mice xenografted with prostate cancer cells and fed RE in their diet showed
a decrease in prostate-specific antigen (PSA) levels (high levels would be suggestive of prostate cancer)
and examination of tissue samples showed decreased androgen receptor and CHOP expression,
indicative of an induction of apoptosis associated with endoplasmic reticular stress [33]. Similarly,
HCT116 colon cancer xenografted mice showed increased Nrf2 and Sestrin-2 expression which are
indicative of endoplasmic reticular stress and can lead to enhanced apoptosis [23]. A significant
decrease in microRNA-15b (miR-15b) plasma levels after administration of RE in colon cancer
xenografted mice suggested circulating miR-15b levels may act as a minimally invasive method
to monitor the antitumor effects of RE in vivo [21]. Furthermore, rats with N-methylnitrosourea
(NMN)-induced colon cancer fed RE, showed significant alterations in cell death modulating proteins
including cytochrome c, programmed cell death protein 4 (PCDP4), carcinoembryonic antigen (CEA)
and colon-cancer specific antigen-4 (CCSA-4) [43]. Sufficient evidence exists to support the potential
use of RE in chemotherapeutics however, it is still not well understood whether the anticancer effects
seen by RE are attributable to individual polyphenols within the extract or rely on the combination of
all the components within the extract combined. The next section of this review explores the role of
two of RE’s main polyphenols, CA and RA, and their potential contribution to RE’s anticancer effects.
Nutrients 2016, 8, 731 12 of 32
6. Anticancer Effects of Carnosic Acid (CA): In Vitro Studies

Treatment of different colon cancer cells with CA resulted in significant inhibition of cell viability
using concentrations ranging from 1 to 400 µM, and having IC50 values in the 20–90 µM range
(Table 7). In addition, CA induced apoptosis and cell cycle arrest in Caco-2 cells [48,49] and inhibited
cell adhesion and migration in Caco-2, HT-29 and LoVo cells [49] by inhibiting activity of the cell
cycle regulator cyclin A [48] and by inhibiting MMP-9, uPA and COX-2 activity, associated with cell
adhesion and migration properties [49]. Similarly, in SW480 colorectal cancer cells with hyperactive
β-catenin which is oncogenic, CA targeted β-catenin for proteasomal degradation and this suggests
a potential for CA to be used as a small molecule oncogenic β-catenin inhibitor [50]. In SLW620
and DLD-1 cells CA inhibited cell viability and this was associated with downregulation of miR-15b
and enhanced GCNT3 activity which are associated with regulation of metabolic related genes [21].
Furthermore, in HT-29 colon cells CA inhibited cell proliferation and enhanced cell cycle arrest, which
was correlated with altered expression of an array of transport and biosynthesis genes and altered
activity of detoxifying enzymes and metabolites. Of note, levels of GSH, an important antioxidant, were
enhanced and levels of N-acetylputrescine, which are toxic in high doses, were decreased [51]. In HT-29
cells co-cultured with 3T3-L1 adipocytes, CA attenuated the negative effects of the adipocytes on the
colon cancer cells by inhibiting triglyceride accumulation and downregulating expression of the Ob-R
receptor [52]. In these cells CA also inhibited cell viability by decreasing phosphorylation of the cell
survival regulators Akt and Bcl-xL and enhancing Bax expression. Furthermore, cell cycle arrest was
induced by inhibition of cyclin D1 and CDK4 [52]. Similarly, a fraction of rosemary extract which was
found to consist mainly of CA (98.7% pure) was tested on HT-29, SW480 and HGUE-C-1 colon cancer
cells and significantly inhibited cell viability. Among several different fractions of the RE that were
tested, the fraction containing CA was found to be among the most active and it was suggested that
synergism between many components of the extract plays a role in rosemary’s anticancer effects [22].
Inhibition of cell proliferation and increased cell cycle arrest by CA in HT-29 cells was found to be
orchestrated by the unfolded protein response and triggered by endoplasmic reticular stress [24]
which can lead to apoptosis and thus destruction of cancerous cells. Enhanced cholesterol and ROS
accumulation in CA treated cancer cells was also shown to contribute to the inhibition of proliferation
seen [24]. Similarly, activity of pro-apoptotic markers including p53, Bax, caspases and PARP were
enhanced and anti-apoptotic markers MDM2, Bcl-2 and Bcl-xL were decreased in HT-29, HCT116 and
SW480 colon cells [53]. Levels of ROS and H2 O2 were increased in vitro in the cell medium [25,53] by
CA which can trigger cellular stress and thus cancer cell death. The signaling molecules STAT3 and
survivin play a key role in regulating cell survival and CA inhibited activity of these molecules in colon
cancer cells [53]. These studies provide strong evidence that CA at relatively low doses (1–100 µM) is
capable of inhibiting colon cancer cell growth and survival by modulating expression of key signaling
molecules and altering cell metabolism.
In breast cancer cells, including MCF-7, MDA-MB-231 and MDA-MB-468, CA inhibited
cell proliferation and enhanced apoptosis at concentrations of 1.5–150 µM [30,54–56] (Table 8).
The inhibitory effects of CA were found to be dependent on increasing levels of the antioxidant
glutathione in breast cancer cells and accordingly, expression of genes involved in glutathione
biosynthesis (CYP4F3, GCLC) and transport (SLC7A11) were significantly increased as well [54].
Importantly, the sensitivity of CA was found to be associated with HER2 expression and thus the
MCF-7 cells were more sensitive to the CA treatment, compared to the triple-negative MDA-MB-468
cell line which does not express HER2 [54]. In the triple negative MDA-MB-361 cell line CA induced
TRAIL-mediated apoptosis through down-regulation of c-FLIP and Bcl-2 expression and through
CHOP-dependent upregulation of DR5, Bim and PUMA expression (ER stress associated proteins) [56]
suggesting that CA is capable of inhibiting breast cancer cell survival through different mechanisms
depending on the mutations that are present.
Nutrients 2016, 8, 731 13 of 32
Table 7. Anticancer effects of Carnosic Acid (CA). In vitro studies: colon cancer.
Cell Type Dose/Duration Findings Mechanism Reference

Caco-2 (Colorectal ↑ cell cycle arrest
1–50 µM CA (48 h) ↓ cyclin A [48]
adenocarcinoma) ↑ cell doubling time
IC50 23 µM
Caco-2 (Colorectal
↑ apoptosis
adenocarcinoma), HT-29
↓ cell adhesion and migration ↓ MMP-9 and uPA activity,
(Colorectal adenocarcinoma), 1–388 µM CA (48 h) [49]
IC50 26.4–92.1 µM COX-2 expression
LoVo (Colorectal
(high in Caco2)
adenocarcinoma)
targets activated β-catenin
SW480 (Colorectal for proteasomal degradation ↓ BCL9-β-catenin
25–100 µM CA (6 h) [50]
adenocarcinoma) and destabilizes interaction
oncogenic β-catenin
SW620 (Colorectal ↑ GCNT3.
2–18 µg/mL
adenocarcinoma), DLD-1 ↓ cell viability ↓ miR-15b gene [21]
(6.02–54.15 µM) CA (48 h)
(Colorectal adenocarcinoma) expression.
↑ GSH levels
Altered expression of
HT-29 (Colorectal 5–35 µg/mL (15–105 µM) ↑ cell cycle arrest
transport and [51]
adenocarcinoma) CA (24–72 h) Alters activity of detoxifying
biosynthesis genes
enzymes and metabolites
↓ N-acetylputrescine
↓ cell viability ↓ p-Akt, cyclin D1,
HT-29 (Colorectal ↑ cell cycle arrest CDK4, Bcl-xL
1–10 µM CA (24–48 h) [52]
adenocarcinoma) ↓ triglyceride accumulation of ↑ Bax expression,
3T3-L1 adipocytes Ob-R expression
HT-29 (Colorectal
adenocarcinoma), SW480 30–60 µg/mL (24–48 h)
(Colorectal adenocarcinoma), CA fraction of RE ↓ cell viability [22]
HGUE-C-1 (98.7% purity)
(Colorectal carcinoma)
↑ UPR
↑ ER-stress
HT-29 12.5 µg/mL (37.6 µM) ↑ cell cycle arrest
↓ cell cycle genes [24]
(Colorectal adenocarcinoma) CA (2–72 h) ↑ cholesterol accumulation
Altered cholesterol-
↑ ROS accumulation
modulating genes
HT-29 (Colorectal ↑ p53, Bax, casp 3, casp 9,
adenocarcinoma), HCT116 PARP cleavage
↓ cell viability
(Colorectal carcinoma), 20–100 µM CA (24 h) ↑ ROS generation [53]
↑ apoptosis
SW480 (Colorectal ↓ MDM2, Bcl-2, Bcl-xL
adenocarcinoma) ↓ survivin, cyclins STAT3
HT-29 (Colorectal 8.3–16.6 µg/mL (25–50 ↑ H2 O2
↓ cell proliferation [25]
adenocarcinoma) µM) CA (24 h) ↑ ROS
MMP-9 (matrix metallopeptidase 9), uPA (urokinase plasminogen activator), COX-2 (cyclooxygenase 2),
BCL9-β (B-cell CLL/lymphoma 9), GCNT3 (glucosaminyl (N-Acetyl) transferase 3), GSH (glutathione),
CDK4 (cyclin-dependent kinase 4), Bcl-xL (B-cell lymphoma-extra large), Bax (Bcl-2-like protein 4), Ob-R
(leptin receptor), ROS (reactive oxygen species), UPR (unfolded protein response), ER (endoplasmic reticulum),
casp (caspase), p53 (tumor protein p53), PARP (poly(ADP-ribose) polymerase), MDM2 (mouse double minute 2
homolog), Bcl-2 (B-cell CLL/lymphoma 2), STAT3 (signal transducer and activator of transcription 3), H2 O2
(hydrogen peroxide).
Inhibition of cell viability by CA was shown in rat insulinoma (RINm5F) and human (MIA-PaCa-2,
PANC-1) pancreatic cancer cells at doses of 6–300 µM [21,27]. In prostate cancer cells, lower
doses of CA (<100 µM) inhibited cell viability and enhanced apoptosis [30,57,58]. Induction of
apoptosis in PC-3 prostate cells was associated with activation of both intrinsic and extrinsic apoptotic
pathways. Inhibition of caspase 8 and 9, Bcl-2, Bid, IAP, p-Akt, p-GSK3 and NF-κB and activation of
caspase 3 and 7, PARP, Bax, cytochrome c and PP2A all contribute to enhanced apoptosis within
these cells [58]. The use of a pan-caspase inhibitor attenuated the apoptotic effects of CA and
provides strong evidence for the involvement of caspases in the apoptotic mechanism of CA in
prostate cancer cells [58]. Low doses of CA both alone and in combination with other phytonutrients
such as curcumin showed potent anticancer effects in LNCaP, PC3 and DU145 prostate cells and
Nutrients 2016, 8, 731 14 of 32
inhibited androgen receptor activity. The inhibition of proliferation of these cells was associated with
an inhibition of the EpRE/ARE antioxidant transcription system and inhibition of PSA secretion [57].
Furthermore, CA inhibited proliferation of A2780 ovarian cancer cells and enhanced the sensitivity
of a resistant A2780CP70 cell line to cisplatin, a potent chemotherapeutic agent [34]. Carnosic acid
has potent anticancer effects on its own but also acts synergistically with other compounds including
phytonutrients and chemotherapeutics and this represents a promising route for future cancer therapies
using combinations of anticancer agents at lower doses.
Table 8. Anticancer effects of Carnosic Acid (CA). In vitro studies: breast, pancreatic, prostate and
ovarian cancer.

MCF-7 (ER+)
(Breast adenocarcinoma), 6.25–50 µg/mL
↓ cell viability [30]
MDA-MB-231 (Breast (18.8–150 µM) CA (48 h)
adenocarcinoma)
MCF-7 ↓ proliferation
↑ CYP4F3, GCLC,
(Breast adenocarcinoma), 0.5–40 µg/mL (1.5–120 µM) ↑ apoptosis
SLC7A11, CDKN1A [54]
MDA-MB-468 CA (6–96 h) ↑ cell cycle arrest IC50:
expression
(Breast adenocarcinoma) 3µg/mL (9 µM) (88 h)
MDA-MB-361
20–60 µM CA (24 h) ↑ apoptosis [55]
↑ TRAIL-mediated
apoptosis
MDA-MB-361 ↓ proliferation
20 µM CA (24 h) ↓ c-FLIP, Bcl-2 [56]
(Breast adenocarcinoma) ↑ apoptosis
↑ DR5, Bim,
PUMA, CHOP
12–100 µg/mL
RINm5F (Insulinoma) ↓ cell viability [27]
(36.1–300 µM) CA (24–48 h)
MIA-PaCa-2 (Pancreatic
2–18 µg/mL (6.02–54.15 µM)
carcinoma), PANC-1 ↓ cell viability [21]
CA (48 h)
(Pancreatic carcinoma)
DU145 (Prostate carcinoma),
6.25–50 µg/mL
PC3 (Prostate ↓ cell viability [30]
(18.8–150 µM) CA (48h)
adenocarcinoma)
↓ casp 8, casp 9, Bcl-2,
Bid, IAP, p-Akt,
PC3 (Prostate ↓ proliferation
20–100 µM CA (0–72 h) p-GSK3, NF-κB [58]
adenocarcinoma) ↑ apoptosis
↑ casp 3, casp 7, PARP
cleavage, Bax, cyt c, PP2A
LNCaP (Prostate carcinoma),
↓ EpRE/ARE
PC3 (Prostate
10 µM CA (72 h) ↓ proliferation transcription system [57]
adenocarcinoma), DU-145
↓ PSA secretion
(Prostate carcinoma)
A2780 (Ovarian carcinoma),
A2780CP70 2.5–10 µg/mL (7.2–30 µM)
Enhanced sensitivity of [34]
(cisplatin-resistant) CA (48 h)
cisplatin-resistant cells
(Ovarian carcinoma)
CYP4F3 (leukotriene-B(4)omega-hydroxylase 2), GCLC (glutamate-cysteine ligase catalytic subunit), SLC7A11
(solute carrier family 7 member 11), CDKN1A (cyclin-dependent kinase inhibitor 1A), TRAIL (TNF-related
apoptosis-inducing ligand), c-FLIP (cellular FLICE (FADD-like-IL-1β-converting enzyme)-inhibiting protein),
DR5 (death receptor 5), Bim (Bcl-2-like protein 11), PUMA (p53 upregulated modulator of apoptosis), CHOP
(C/EBP homologous protein), casp (caspase), Bcl-2 (B-cell CLL/lymphoma 2), Bid (BH3 interacting-domain),
IAP (inhibitor of apoptosis), p-Akt (phosphorylated protein kinase B), p-GSK3 (phosphorylated glycogen
synthase kinase 3), NF-κB (nuclear factor kappa B), PARP (poly (ADP-ribose) polymerase), Bax (Bcl-2-like
protein 4), cyt c (cytochrome c), PP2A (protein phosphatase 2A), EpRE (electrophile responsive element), ARE
(antioxidant response element), PSA (prostate specific antigen).
In Hep-3B, HepG2 and SK-HEP1 human liver cancer cells, CA inhibited cell viability and
enhanced apoptosis [30,55,56,59] (Table 9). In Hep-G2 cells the formation of autophagic vacuoles and
autolysosomes contributed to enhanced cell death by CA and this was induced through inhibition of the
Akt/mTOR cell survival pathway [59]. Furthermore, in SK-HEP1 cells CA induced TRAIL-mediated
Nutrients 2016, 8, 731 15 of 32
apoptosis by altering apoptotic markers such as c-FLIP, Bcl-2, DR5, Bim, PUMA and CHOP [56].
Rat liver clone 9 cells are often used as a model for screening hepatotoxicity and CA was found to
enhance activity of enhancer element GPEI which regulates the pi class of glutathione S-transferase
and modulates antioxidant and detoxification systems within the cell [60]. CA was found to exert
a protective effect in these non-cancerous liver cells which was modulated by the Nrf2/p38 MAPK
signaling pathway [60,61]. Furthermore, CA inhibited viability of small-cell lung cancer NCI-H82
cells [30].
Table 9. Anticancer effects of Carnosic Acid (CA). In vitro studies: liver, lung, skin and kidney cancer.

Hep-3B 6.25–50 µg/mL
(Hepatocellular carcinoma) (18.8–150 µM) CA (48 h)
↓ proliferation
HepG2 ↑ apoptosis ↑ LC-3
20–100 µM for (12–48 h) [59]
(Hepatocellular carcinoma) ↑ autophagic vacuoles and ↓ p-Akt, p-mTOR
autolysosomes
SK-HEP1
20–60 µM CA (24 h) ↑ apoptosis [55]
↑ TRAIL-mediated
SK-HEP1 ↓ proliferation apoptosis
20 µM CA (24 h) [56]
(Hepatocellular carcinoma) ↑ apoptosis ↓ c-FLIP, Bcl-2
↑ DR5, Bim, PUMA, CHOP
↑ reporter activity of enhancer ↑ GSTP expression
Rat clone 9
1–20 µM CA (24 h) element GPEI ↑ Nrf2 translocation [60]
(Normal rat liver)
↑ detoxification systems ↑ p38
↑ NQO1
Rat clone 9 ↑ Nrf2
1–20 µM CA (0–24 h) ↓ cell survival [61]
(Normal rat liver) ↑ p-p38
↑ p-ERK
NCI-H82 6.25–50 µg/mL
(Lung carcinoma; SCLC) (18.8–150 µM) CA (48 h)
↑ apoptosis
HT-1080 (Fibrosarcoma) 25–100 µM CA (4–72 h) [62]
↑ chromatin condensation and
DNA fragmentation IC50 9 µM
BAEC Aortic endothelial ↓ cell survival
↓ MMP-2
cells), HUVEC ↑ apoptosis
25–100 µM CA (4–72 h) ↓ endothelial cell [62]
(Umbilical vein ↑ cell cycle arrest
tubulogenesis.
endothelial cells) ↓ migration IC50 36µM
↓ cell migration and adhesion
↓ MMP-9, TIMP-1, uPA,
Suppressed mesenchymal
B16F10 (Skin melanoma) 2.5–10 µM CA (12 h) VCAM-1 [63]
markers
↓ p-Src, p-FAK, p-Akt
Induced epithelial markers
Caki (Kidney clear cell ↑ apoptosis Promotes ROS ↑ PARP cleavage, casp 3,
20–60 µM CA (24 h) [55]
carcinoma) production ATF4, CHOP
Caki (Kidney clear cell
↑ TRAIL-mediated
carcinoma), AHCN
↓ proliferation apoptosis
(Kidney renal cell 20 µM CA (24 h) [56]
↑ apoptosis ↓ c-FLIP, Bcl-2
adenocarcinoma), A498
↑ DR5, Bim, PUMA, CHOP
(Kidney carcinoma)
LC3 (light chain 3), p-mTOR (phosphorylated mammalian target of rapamycin), TRAIL (TNF-regulated
apoptosis-inducing ligand), c-FLIP (cellular FLICE (FADD-like-IL-1β-converting enzyme)-inhibiting protein),
Bcl-2 (B-cell CLL/lymphoma 2), DR5 (death receptor 5), Bim (Bcl-2-like protein 11), PUMA (p53 upregulated
modulator of apoptosis), CHOP (C/EBP homologous protein), GSTP (Glutathione S-transferase P), Nrf2 (nuclear
factor E2-related factor-2), NQO1 (NAD(P)H-quinone oxidoreductase 1), p-ERK (phosphorylated extracellular
signal-regulated kinases), MMP-2 (matrix metalloproteinase-2), MMP-9 (matrix metallopeptidase-9), TIMP-1
(TIMP metallopeptidase inhibitor 1), uPA (urokinase plasminogen activator), VCAM-1 (vascular cell adhesion
protein 1), p-Src (proto-oncogene tyrosine-protein kinase Src), p-FAK (phosphorylated focal adhesion kinase),
PARP (poly(ADP-ribose)polymerase), casp (caspase).
In several models of skin cancer, including HT-1080, BEAC, HUVEC and B16F10 cells, CA
inhibited cell survival, cell migration and cell adhesion, enhanced apoptosis and induced cell cycle
arrest [62,63] (Table 9). Chromatin condensation and DNA fragmentation were seen in HT-1080 cells
Nutrients 2016, 8, 731 16 of 32
which lead to apoptosis [62]. In human umbilical and bovine aortic endothelial cell lines, CA inhibited
tubulogenesis and MMP-2 expression suggesting anti-angiogenic properties of CA which would be
beneficial in anticancer therapies [62]. Inhibition of the epithelial-mesenchymal transition in B16F10
melanoma cells suggests a possible mechanism for the inhibition of cell migration by CA. Inhibition
of cell migration markers MMP-9, TIMP-1, uPA and VCAM-1 was seen in this cell line using low
doses of CA (10 µM). Inhibition of phosphorylation of signaling molecules Akt, FAK and Src were
also associated with inhibition of the epithelial-mesenchymal transition and cell migration in B16F10
cells [63]. In Caki, kidney cancer cells, CA induced apoptosis through ROS-mediated endoplasmic
reticular stress. Activity of apoptotic markers PARP, caspase 3, ATF4 and CHOP was increased in these
cells [55]. Similarly, TRAIL-mediated apoptosis was induced in Caki, AHCN and A498 kidney cells
through modulation of endoplasmic reticular stress related proteins c-FLIP, Bcl-2, DR5, Bim, PUMA
and CHOP [56].
In T98G glioblastoma cells CA promotes production of nerve growth factor and this was found
to be regulated by the Nrf2 signaling pathway [64,65] (Table 10). Nerve growth factor is involved in
the regulation of growth and the maintenance and survival of certain target neurons, and thus can
act to protect neural cells from toxic agents that may cause cancer. In IMR-32 neuroblastoma cells CA
induced apoptosis by activation of caspases, PARP and the p38 MAPK pathway and inhibited cell
viability, which was associated with decreased ERK activation [66]. Interestingly however, in SH-SY5Y
neuroblastoma cells CA attenuated apoptosis induced by the neurotoxic compounds methylglyoxal
and amyloid β, exerting a cytoprotective effect [67,68]. This protective effect was associated with
increased activation of PI3K/Akt signaling, inhibition of cytochrome c release and inhibition of caspase
cascades which results in a pro-survival effect on the cell [36,67]. Similarly, in U373MG astrocytoma
cells CA inhibited amyloid β peptide production and release and this was associated, at least partially,
with activation of the α-secretase TACE/ADAM17 [69]. The use of CA may have potential in the
prevention of amyloid β-mediated diseases. Furthermore, in GBM glioblastoma cells, CA promoted
apoptosis by inducing cell cycle arrest and degradation of cyclin B1, RB, SOx2 and GFAP, molecules
involved in cell survival and maturation processes [70].
Table 10. Anticancer effects of Carnosic Acid (CA). In vitro studies: brain and neural cancer.

T98G (Glioblastoma) 5–100 µM CA (0–48 h) ↑ NGF synthesis [64]
↑ NGF synthesis
T98G (Glioblastoma) 2–50 µM CA (24 h) [65]
↑ Nrf2, HO-1, TXNRD1
↓ cell viability
IMR-32 ↑ casp 3, casp 9, PARP, p-p38
5–40 µM CA (0–48 h) ↑ apoptosis [66]
(Neuroblastoma) ↓ p-ERK
↑ ROS generation
U373MG ↑ α-secretase
50 µM CA (8 h) ↓ amyloid beta peptide release [69]
(Glioblastoma) TACE/ADAM17
↑ antioxidant defense ↑ PI3K/Akt
SH-SY5Y
1 µM CA (12 h) ↑ detoxification systems ↓ cytochrome c release [67]
(Neuroblastoma)
Blocked activation of apoptosis ↓ caspase cascade
SH-SY5Y
10 µM CA (1 h) ↓ apoptosis ↓ caspase cascade [68]
(Neuroblastoma)
↓ CDK activity
↓ cell survival ↓ cyclin B1
GBM (Glioblastoma) 17.5–40 µM CA (48 h) ↑ cell cycle arrest ↓ RB [70]
↑ apoptosis ↓ SOX2
↓ GFAP
NGF (nerve growth factor), Nrf2 (nuclear factor E2-related factor 2), HO-1 (heme oxygenase-1), TXNRD1
(thioredoxin reductase 1), casp (caspase), PARP (poly(ADP-ribose)polymerase), p-ERK (phosphorylated
extracellular signal-regulated kinases), TACE (TNF-α converting enzyme), ADAM17 (ADAM metallopeptidase
domain 17), PI3K (phosphatidylinositol-4,5-bisphosphate 3-kinase), Akt (protein kinase B), cyt c (cytochrome
c), CDK (cyclin dependent kinase), RB (retinoblastoma), SOX2 (sex determining region Y-box 2), GFAP (glial
fibrillary acidic protein).
Nutrients 2016, 8, 731 17 of 32
Leukemia is a cancer that usually develops in the bone marrow and results in a high number of
white blood cells that are not fully developed being released into the bloodstream. Most treatment
options for leukemia involve agents that promote the differentiation of these immature white blood
cells into mature, differentiated cells. Unfortunately, there are many side effects associated with higher
doses of these differentiating agents and strategies are required to lower the dose necessary to see
anticancer effects. One such agent which is used is 1α25-dihydroxyviatmin D (1,25D). Many studies
have found that low doses of CA (5–10 µM) are able to potentiate the pro-differentiation effects of
1,25D and help sensitize leukemia cells including human HL-60, U937, MOLM-13 and mouse WEHI-3B
cells [40,41,71–78] to its anticancer effects (Table 11). Furthermore, CA inhibited cell viability and
induced apoptosis and cell cycle arrest in these cells using a multitude of different strategies. In
HL-60 cells, CA enhanced expression of the vitamin D and retinoic acid receptors thus, enhancing the
sensitivity of cells to 1,25D [71], and enhanced expression of cell cycle regulators p21Waf1 , p27Kip1 which
may have tumor suppressor functions [72]. Carnosic acid also increased levels of the antioxidant GSH
and phase II enzyme NADP(H)-quinone reductase which help protect cells from chemically-induced
carcinogenesis, and enhanced signaling through MAPK pathways including ERK and JNK which
are involved in the proliferation and differentiation of cells [40,73–75,79]. In K562 leukemia cells CA
inhibited cell viability and sensitized resistant cells to adriamycin, a chemotherapeutic agent [30,80].
Similarly, CA enhanced the activity of doxercaliferol, an agent which helps prevent the common
problem of calcification associated with administration of vitamin D derivatives such as 1,25D, and
decreased levels of microRNA181a which are linked to cell proliferation [81]. Antioxidant effects were
also produced by CA in U937, HL-60 and NB4 leukemic cells which exhibited increased GSH and
NADPH levels and CA ameliorated arsenic trioxide-induced cytotoxic effects [79]. Activation of the
Nrf2/ARE signalling pathway which can alter cell survival was also seen [77,79]. The authors suggest
that the Nrf2/ARE pathway likely plays an important role in the cooperative induction of leukemia cell
differentiation by 1,25D and CA [77]. Importantly, in HL-60 cells CA increased PTEN expression and
caspase cleavage and inhibited phosphorylation of Bad and Akt which are associated with enhanced
apoptosis [62,82]. The strong inhibitory effects of CA on the PTEN/Akt survival pathway make it a
good candidate to be combined with other therapies for leukemia treatment.
Table 11. Anticancer effects of Carnosic Acid (CA). In vitro studies: leukemia.

CA potentiated effects of 1,25D
HL-60 (Myeloid ↑ differentiation ↑ vitamin D receptor,
10 µM CA (0–48 h) [71]
leukemia) ↓ proliferation retinoic acid receptor
HL-60 (Myeloid
↑ differentiation
leukemia), U937 2.5–10 µM CA (0–48 h) ↑ p21Waf1 , p27Kip1 [72]
↓ proliferation
(Myeloid leukemia)
↑ cell cycle arrest IC50 6–7µM
HL-60-G ↑ GSH
10 µM CA (0–48 h) ↑ differentiation [73]
(Myeloid leukemia) ↑ Raf/MAPK/ERK, AP-1
↓ ROS
HL-60 CA potentiated effects of 1,25D
10 µM CA (0–72 h) ↑ JNK pathway [74]
(Myeloid leukemia) ↑ differentiation
WEHI-3B (Murine
myeloid leukemia),
↑ differentiation
HL-60 (Myeloid 10 µM CA (0–96 h) [41]
↓ proliferation
leukemia), U937
(Myeloid leukemia)
↓ ROS
WEHI-3B D (Murine ↑ cell differentiation
10 µM CA (48–96 h) ↑ NADP(H)-quinone [40]
myeloid leukemia) ↓ cell viability
reductase
↓ cell viability CA sensitized
K562 (Myeloid leukemia) 2.5–50 µM CA (24–72 h) [80]
resistant cells to Adriamycin
Nutrients 2016, 8, 731 18 of 32
Table 11. Cont.

HL-60G (Myeloid
CA potentiated effects of 1,25-D ↑ JNK1, c-jun-ATF2,
leukemia), HL-60-40AF 10 µM CA (0–48 h) [75]
↑ differentiation C/EBP
(Myeloid leukemia)
K-562 6.25–50 µg/mL
(Myeloid leukemia) (18.8– µM) CA (48 h)
U937 CA potentiated effects of 1,25-D
10 µM CA (96 h) ↑ Nrf2, ARE, NADPH, [77]
(Myeloid leukemia) ↑ differentiation
HL-60 Enhances activity of 1,25D
(Myeloid leukemia), ↑ cell cycle arrest Induces
10 µM CA (48 h) ↑ HPK1 [76]
U937 differentiation Sensitizes 1,25D
(Myeloid leukemia) resistant cells
Enhances activity of
HL-60
doxercalciferol
(Myeloid leukemia), 10 µM CA (48 h) ↓ microRNA181a [81]
↑ cell cycle arrest Induces
U937
differentiation
↓ viability ↑ p27, cleaved casp 9,
HL-60
5–25 µM CA (24–72 h) ↑ apoptosis PTEN expression [82]
(Myeloid leukemia)
↑ cell cycle arrest ↓ p-BAD, p-Akt
↓ cell survival
HL-60
25–100 µM CA (4–72 h) ↑ apoptosis ↑ casp 3 [62]
(Myeloid leukemia)
↑ cell cycle arrest IC50 5.7 µM
HL-60 (Myeloid
leukemia), U937
CA potentiated effects of 1,25-D
(Myeloid leukemia), 10 µM CA (96 h) [78]
↑ differentiation
MOLM-13 (Acute
monocytic leukemia)
Ameliorates arsenic
NB4 (Human ↑ GSH levels
5 µM CA (24 h) trioxide-induced [79]
promyelocytic leukemia) Activation of Nrf2
cytotoxic effects
1,25-D (1α25-dihydroxyviatminD), GSH (glutathione), Raf (rapidly accelerated fibrosarcoma), MAPK
(mitogen-activated protein kinase), ERK (extracellular signal-regulated kinases), AP-1 (activator protein 1),
JNK (c-jun N-terminal kinases), ROS (reactive oxygen species), c-jun (v-jun sarcoma virus 17 oncogene), ATF2
(activating transcription factor 2), Nrf2 (nuclear factor E2-regulated factor-2), ARE (antioxidant response
element), HPK1 (hematopoietic progenitor kinase 1), casp (caspase), PTEN (phosphatase and tensin homolog).
7. Anticancer Effects of Carnosic Acid (CA): In Vivo Animal Studies

The above studies in vitro provide strong evidence for the anticancer effects if CA in various
cancer cell lines. Several studies using animal models have also explored the effects of CA in vivo
and found significant anticancer effects which supports future research exploring the anticancer
mechanisms of CA in both animal and human models (Table 12). In DMBA-induced models of oral
cancer using hamsters, it was shown that using 10 mg/kg/day CA administered orally for 14 weeks,
caused the number of tumors on the animals to significantly decrease. Furthermore, expression
of detoxification enzymes was enhanced [83], markers of apoptosis including p53, Bax, Bcl-2 and
caspases were increased [84], and regulators of cell growth including COX-2, c-fos, KF-κB and cyclin
D1 were decreased [84]. Using the same hamster model, 750 µg CA dissolved in 0.1mL saline (20 µM)
administered daily for 11 weeks significantly slowed the progression of lesions and oral cancer
development [85]. In mice xenografted with prostate samples from human biopsies, 100 mg CA
dissolved in 100 µL of cottonseed oil administered daily for 25 days decreased tumor growth [86].
Azoxymethane was used to induce colon cancer in mice and 0.01%–0.02% CA fed with a high fat
(45%) diet for 11 weeks decreased both tumor size and number of tumors, and modulated signaling
molecules involved in cell metabolism and cell growth [52]. Serum samples taken from the mice after
treatment showed decreased levels of insulin, leptin and IGF-1 and analysis of tissue samples showed
a decrease in the associated insulin and leptin receptors, as well as decreased activity of ERK and
expression of cyclin D1 and Bcl-xL which regulate cell survival [52]. In K562 leukemia inoculated mice
fed 1% CA with standard powder diet, there was a decrease in the number of leukemic cells which was
Nutrients 2016, 8, 731 19 of 32
partially attributed to enhanced apoptosis [87]. Furthermore, survival time of the animals increased
significantly [87]. Overall, CA shows significant anticancer effects in mouse and hamster models of
several types of cancer and this evidence provide support of its potential to be used against cancer
in humans.
Table 12. Anticancer effects of Carnosic Acid (CA). In vivo studies.
Animal Model Dose/Duration Findings Mechanism Reference

↓ # of tumors Anti-lipid
DMBA-induced oral 10 mg/kg/day CA
peroxidative function [83]
cancer-hamster (14 weeks)
↑ detoxification enzymes
↑ p53, Bax, Bcl-2,
DMBA-induced oral 10 mg/kg/day CA orally casp 3, casp 9
↓ # of tumors [84]
cancer-hamster for (14 weeks) ↓ COX-2, c-fos, NF-κB,
cyclin D1
Human prostate 100 mg/mouse dissolved
biopsies xenografted in 100 µL cottonseed oil ↓ tumor growth [86]
into mice daily (25 days)
750 µg CA dissolved in
DMBA-induced oral ↓ progression of cancer and
0.1 mL saline (20 µM) [85]
cancer-hamster development of lesions
daily for (11 weeks)
↓ insulin, leptin and IGF-1
serum levels compared to
0.01%–0.02% CA fed with
AOM-induced colon ↓ # of tumors mice fed HFD alone
a high fat (45%) diet for [52]
cancer-mice ↓ tumor size ↓ insulin receptor, leptin
(11 weeks)
receptor, p-ERK, cyclin D1,
Bcl-xL expression
1% (v/v) CA with ↓ # of leukemia cells
K562 leukemia
standard powdered rodent ↑ apoptotic cells [87]
inoculated mouse
diet Ad libitum ↑ survival time
Bax (Bcl-2-like protein 4), Bcl-2 (B-cell CLL/lymphoma 2), casp (caspase), COX2 (cyclooxygenase 2), NF-κB
(nuclear factor kappa B), IGF-1 (insulin-like growth factor 1), HFD (high fat diet), p-ERK (phosphorylated
extracellular signal-regulated kinase), Bcl-xL (B-cell lymphoma-extra large), # (number).
8. Anticancer Effects of Rosmarinic Acid (RA): In Vitro Studies

Treatment of HT29 colon cancer cells with RA (5–20 µM) lead to a reduction in COX2 promoter
activity and COX2 protein levels [88] (Table 13). In HCT15 and CO115 colon cancer cells, RA
(10–100 µM) induced apoptosis and decreased levels of phosphorylated-ERK which regulates cell
proliferation [89]. Rosmarinic acid (55–832.6 µM) decreased ROS levels which was associated with
decreased migration and adhesion rates in Ls174-T colon cells [90]. Furthermore, treatment of
CO115 cells with RA (50 µM) protected against BCNU-induced DNA damage, suggesting potential
chemopreventive effects [91]. Treatment of MCF-7 and MDA-MB-231 breast cancer cells with RA
(0–300 µM) decreased cell viability [30,92–94] (Table 13). Rosmarinic acid decreased methyltransferase
activity, which inhibits hyper-methylation of DNA, associated with disease [93], and sensitized a
resistant cell line (MCF-7/Adr) to the chemotherapeutic agent Adriamycin [94].
In DU145 and PC3 prostate cancer cells RA (17.3–138.8 µM) decreased cell viability [30] and in
A2780 and A2790CP70 ovarian cancer cells RA (6.9–27.8 µM) lead to a reduction in cell proliferation
and increased the sensitivity of cisplatin-resistant cells [34] (Table 13). In SCG7901/Adr gastric cancer
cells, RA (0.096–60 µM) was found to decrease cell viability, drug resistance, expression and activity of
p-glycoprotein [95]. Furthermore, treatment of MKN45 gastric cancer cells with RA (200–300 µM) lead
to a decrease in cell viability, the Warburg effect/glucose uptake and pro-inflammatory cytokines [96].
In B16 melanoma cells, RA (1–100 µM) was found to increase melanin content, tyrosinase expression
and CREB phosphorylation [97].
Nutrients 2016, 8, 731 20 of 32
Table 13. Anticancer effects of Rosmarinic Acid (RA). In vitro studies: colon, breast, prostate, ovarian,
gastric and skin cancer.
Cell Type Dose and Duration Findings Mechanisms Reference

HT-29 (Colorectal ↓ TPA induced COX2
5–20 µM RA (1 h) ↓ COX2 protein levels [88]
adenocarcinoma) promoter activity
HCT15 (Colorectal
↑ apoptosis of HCT15
adenocarcinoma), ↓ p-ERK levels in
10–100 µM RA (48 h) (50 µM) and CO115 [89]
CO115 (Colorectal HCT15 cells
(100 µM)
carcinoma)
Ls174-T (Colorectal 20–300 µg/mL ↓ migration rate
↓ ROS [90]
adenocarcinoma) (55.5–832.6 µM) RA (24 h) ↓ adhesion IC50 70 µg/mL
CO115 (Colorectal ↓ BCNU-induced DNA
50 µM RA (24 h) [91]
carcinoma) damage
MCF-7 (Breast
60 µM RA (24 h) ↓ cell viability [92]
adenocarcinoma)
MCF7 (Breast ↓ DNA methyltransferase
2–200 µM RA (72 h) [93]
adenocarcinoma) activity
MCF-7 (ER+) (Breast
adenocarcinoma), 6.25–50 µg/mL
MDA-MB-231 (Breast (17.3–138.8 µM) RA (48 h)
adenocarcinoma)
MCF-7/Adr (Breast
0.08–10 mM RA EC values:
adenocarcinoma), 0.08–0.32 mM RA effective
0.74 mM (in wt) and [94]
MCF-7/wt (Breast ↑ cytotoxicity to MCF-7 cells
0.81 mM (in Adr resistant)
adenocarcinoma)
DU145 (Prostate
carcinoma), PC3 6.25–50 µg/mL
(Prostate (17.3–138.8 µM) RA (48h)
adenocarcinoma)
A2780
↓ cell proliferation Enhanced
(Ovarian carcinoma), 2.5–10 µg/mL
sensitivity of [34]
A2780CP70 (6.9–27.8 µM) RA (48 h)
cisplatin-resistant cells
(Ovarian carcinoma)
↓ expression of
SGC7901/Adr ↓ cell viability Reversed
0.096–60 µM RA (48 h) p-glycoprotein [95]
(Gastric carcinoma) drug resistance
↓ activity of p-glycoprotein
↓ glucose uptake
MKN45 ↓ cell viability
200–300 µM RA ↓ pro-inflammatory [96]
(Gastric carcinoma) ↓ Warburg effect
cytokines (IL-6 and STAT3)
↑ melanin content ↑ phosphorylation
B16 (Skin melanoma) 1–100 µM RA (48 h) [97]
↑ tyrosinase expression of CREB
TPA (12-O-tetradecanoylphorbol-13-acetate), COX2 (cyclooxygenase 2), ERK (extracellular signal-regulated
kinases), ROS (reactive oxygen species), BCNU (1,3-bis-(2-chloroethyl)-1-nitosourea), IL-6 (interleukin-6), STAT3
(signal transducer and activator of transcription 3) CREB (cAMP response element-binding protein) wt (wild
type), Adr (Adriamycin).
Treatment of HepG2 liver cancer cells with RA (25–250 µM) decreased ochratoxin and
aflatoxin-mediated cell damage, apoptosis, ROS levels and caspase 3 activation [98] (Table 14),
suggesting that RA can exert protective effects and prevent cytotoxicity induced by toxic agents.
Alternatively, in HepG2 cells without the presence of cytotoxic agents, RA (13.9 and 27.8 µM) lead
to an increase in apoptosis, which was associated with an increase in caspase 8, NFBIA, TNFSF9
and Jun mRNA and a decrease in Bcl-2 mRNA levels [99]. Thus, RA has several potential anticancer
mechanisms in liver cells. In Hep-3B liver cancer cells, RA (17.3–138.8 µM) was found to decrease cell
viability [30], while treatment of HepG2 liver cancer cells with RA (20–80 µM) showed no significant
changes to cell viability but an increase in Nrf2 nuclear translocation, ARE-luciferin activity, MRP2
levels, intracellular ATP levels and efflux of p-glycoprotein was seen [100]. In NCI-H82 and A549
lung cancer cells RA (10–500 µM) decreased cell growth [30,101] which was associated with decreased
hCOX2 activity, suggesting an anti-inflammatory role of RA [101].
Nutrients 2016, 8, 731 21 of 32
Table 14. Anticancer effects of Rosmarinic Acid (RA). In vitro studies: liver and lung cancer.

↓ OTA- and AFB-induced
cell damage and apoptosis
HepG2 (Hepatocellular ↓ ROS production
25–250 µM RA (24 h) ↓ DNA and protein [98]
carcinoma) ↓ capase-3 activation
synthesis inhibition induced
by OTA- and AFB-
↑ casp 8, NFBIA, TNFSF9
HepG2 (Hepatocellular 5–10 µg/mL
↑ apoptosis and Jun mRNA [99]
carcinoma) (13.9–27.8 µM) RA (72 h)
↓ Bcl-2 mRNA expression
HepG2 (Hepatocellular
60 µM RA (24 h) ↓ cell viability [92]
carcinoma)
Hep-3B (Hepatocellular 6.25–50 µg/mL
carcinoma) (17.3–138.8 µM) RA (48 h)
↑ translocation of Nrf2
↑ ARE-luciferin activity
HepG2 (Hepatocellular 20–80 µM RA
↔cell viability ↑ efflux of p-glycoprotein [100]
carcinoma) (24 h or 4 days)
↑ MRP2
↑ intracellular ATP
NCI-H82 (Lung 6.25–50 µg/mL
carcinoma; SCLC) (17.3–138.8 µM) RA (48 h)
A549 (Lung 10–500 µM RA (48 h)
↓ cell proliferation ↓ hCOX2 activity [101]
adenocarcinoma) IC50 198.12
OTA (ochratoxin), AFB (Aflatoxin), ROS (reactive oxygen species), casp (caspase), NFBIA (nuclear factor
of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha), TNFSF9 (tumor necrosis factor ligand
superfamily-member 9), Jun (v-jun sarcoma virus 17 oncogene), Bcl-2 (B-cell CLL/lymphoma 2), Nrf2 (nuclear
factor E2-related factor-2), ARE (antioxidant response element), MRP2 (multidrug resistance-associated
protein 2), ATP (adenosine triphosphate), hCOX2 (human cyclooxygenase 2).
Treatment of K562 leukemia cells with RA inhibited cell viability [30] and reversed the induction
of hyperosmosis-induced apoptosis and associated ROS/RNS production [102] (Table 15). In U937
leukemia cells, RA (60 µM) enhanced TNF-α induced apoptosis and decreased TNF-α induced-NF-κB
activation and ROS production [92]. Surprisingly AKT1 and ERK2 levels, which regulate cell survival,
were not affected by RA treatment in U937 or K562 cells [42]. Rosmarinic acid (40 µM) increased
macrophage differentiation induced by ATRA which was mediated by an increase in CD11b expression
on the cell surface [103]. In HL-60 leukemia cells, RA (50–150 µM) inhibited cell growth and induced
apoptosis, which was associated with decreased dNTP levels [104]. CCRF-CEM, CEM/ADR5000
leukemia cells treated with RA (3–100 µM) developed increased cytotoxicity, apoptosis, necrosis, cell
cycle arrest and caspase-independent apoptosis which was mediated by increased PARP cleavage
and blockage of p65 nuclear translocation [105]. In agreement with other studies, RA (0.07–2.2 mM)
exerted DNA protective and anti-carcinogenic effects in HL-60 leukemia cells [106].
Table 15. Anticancer effects of Rosmarinic Acid (RA). In vitro studies: leukemia.

↓ hyperosmotic-mediated
K562 (Myeloid leukemia) 25 µM RA (1 h) ROS/RNS production [102]
and apoptosis
↓ NF-κB activation
U937
60 µM RA (24 h) ↑ TNF-α induced apoptosis ↓ ROS production [92]
(Myeloid leukemia)
↑ caspases
6.25–50 µg/mL
K562 (Myeloid leukemia) ↓ cell viability [30]
(17.3–138.8 µM) (48 h)
K562 (Myeloid
↔ AKT1
leukemia), U937 0.2 mM RA (48 h) Not tested on proliferation [42]
↔ ERK2
(Myeloid leukemia)
NB4 (Human ↑ ATRA-induced
40 µM RA (72 h) ↑ expression of CD11b [103]
promyelocytic leukemia) macrophage differentiation
Nutrients 2016, 8, 731 22 of 32
Table 15. Cont.

↓ cell growth
↑ apoptosis
HL-60 (Myeloid leukemia) 50–150 µM RA (24–72 h) ↓ dNTP levels [104]
IC50 147 µM (24 h), 74 µM
(48 h), 69 µM (72 h)
↑ cytotoxicity
CCRF-CEM
↑ apoptosis and necrosis ↑ PARP-cleavage Blocked
(Lymphoblastic leukemia),
3–100 µM RA (72 h) ↑ cell cycle arrest p65 nuclear translocation [105]
CEM/ADR5000
↑ caspase-independent from the cytosol
(Lymphoblastic leukemia)
apoptosis
DNA protection and
HL-60 (Myeloid leukemia) 0.07–2.2 mM RA (72 h) [106]
anticarcinogenic effects
ROS (reactive oxygen species), RNS (reactive nitrogen species), TNF-α (tumor necrosis factor-alpha), NF-κB
(nuclear factor kappa-light-chain-enhancer of activated B cells), Akt (protein kinase B), ERK (extracellular
signal-regulated kinases), ATRA (all-trans retinoic acid), dNTP (deoxy-nucleoside triphosphate), PARP
(poly(ADP-ribose) polymerase).
9. Anticancer Effects of Rosmarinic Acid (RA): In Vivo Animal Studies

Apart from the in vitro studies using different cancer cell lines, several studies using RA in animal
cancer models have been performed. Administration of 0.25–1.35 mg of RA (30 min) prior to TPA
treatment was found to decrease myeloperoxidase activity and COX2 induction in mice [107] (Table 16).
Using 1–4 mg/kg RA (20 days) in Lewis lung carcinoma xenografted mice lead to decreased tumor
growth [90] and 100 mg/kg RA (14 weeks) reduced DMBA-induced tumor formation in the buccal
pouches of hamsters [108]. Administration of 360 mg/kg RA from weeks 4 to 12 of the animal’s life
decreased the frequency of large adenomas in mice [109]. Rats given 2.5–10 mg/kg RA for 16 weeks,
showed a decrease in development of DMH-induced aberrant crypt foci by decreasing DMH-induced
elevation of bacterial enzymes [110]. Administration of 100 mg/kg RA 1 week before DMBA treatment
in mice decreased skin tumors by increasing the levels of phase I (cyt p450) and phase II (GST, GR, GSH)
detoxification agents and restoring levels of caspase 3, caspase 9, p53 and Bcl-2 [111]. Venkatachalam,
et al. found that 2.5, 5 and 10 mg/kg RA given to rats for 4 weeks, decreased DMH-induced colon
tumor formation, number of polyps, antioxidant status, CYP450 content, PNPH activity and reversed
the markers of oxidative stress [112]. Hamsters given 1.3 mg/mL RA for 2 weeks were found to have
a decreased incidence of tumors induced by DMBA, decreased tumor grade scoring and increased
tumor differentiation [113]. Rosmarinic acid administered at 2 mg/kg for 14 days to mice had an
anti-Warburg effect, mediated through decreased glucose uptake [96]. Furthermore, administration of
5 mg/kg RA for 30 weeks was found to decrease DMH-induced colon tumor formation in rats through
decreased TNF-α, IL-6 and COX2 levels [110]. Taken together, these studies provide evidence for RA’s
anticancer effects in animal models and suggest several mechanisms which may be responsible for the
inhibition of tumor growth and progression.
Table 16. Anticancer effects of Rosmarinic Acid (RA). In vivo studies.
Animal Model Dose and Duration Findings Mechanisms Reference

0.25, 0.5, 1.0 and
Seven-Nine week old male 1.35 mg/mouse
↓ myeloperoxidase activity ↓ COX2 induction [107]
Balb/c mice (30 months) before
TPA treatment
C57BL/6 mice implanted 1, 2 and 4 mg/kg RA
↓ tumor growth [90]
with Lewis lung carcinoma (20 days)
Completely prevented
100 mg/kg RA ↓ p53
Golden Syrian hamsters tumor formation in [108]
(14 weeks) ↓ Bcl-2
DMBA-treated hamsters
C57BL/6J Min/+ (ApcMin ) ↓ the frequency of ↑ levels of parent
360 mg/kg RA (8 weeks) [109]
mice large adenomas compound in plasma
Nutrients 2016, 8, 731 23 of 32
Table 16. Cont.
Animal Model Dose and Duration Findings Mechanisms Reference

DMH induced colon 2.5–10 mg/kg RA
↓ DMH induced aberrant ↓ DMH induced increase
cancer (Albino Wistar (16 weeks) through [110]
crypt foci in bacterial enzymes
male rats) intragastric intubation
↑ status of phase I (cyt
p450) detoxification agents
100 mg/kg RA ↑ status of phase II (GST,
DMBA induced skin
administered (1 weeks) ↓ skin tumors GR, GSH) detoxification [111]
cancer (Swiss albino mice)
before DMBA treatment agents. Restored activity
levels of casp 3, casp 9, p53
and Bcl-2.
↓ DMH induced aberrant
crypt foci, number of polyps,
DMH induced colon 2.5, 5 and 10 mg/kg RA reversed the markers of
[112]
cancer (Male Wistar rats) (4 weeks) oxidative stress, antioxidant
status, CYP450 content and
PNPH activity
↓ incidence of tumors
↑ differentiation
Five month old Syrian 1.3 mg/mL RA (2 weeks)
↓ scores in the tumor [113]
hamsters pretreatment
invasion front grading
system.
5 week old male nude
Balb/c mice incubated
2 mg/kg RA via celiac
sub-cutaneously with ↓ Warburg effect ↓ glucose uptake [114]
injection daily (14 days)
MKN45 cells into
their flanks.
↓ TNF-α
DMH induced colon 5 mg/kg RA orally ↓ DMH induced colon
↓ IL-6 [96]
cancer (Male Wistar rats) (30 weeks) tumor formation
↓ COX2
TPA (12-O-tetradecanoylpheorbol-13-acetate), COX2 (cyclooxygenase 2), DMBA
(7,12-dimethylbenz(a)anthracene), DMH (1,2-dimethylhydrazine), p53 (tumor protein p53), casp (caspase), Bcl-2
(B-cell CLL/lymphoma 2), CYP450 (cytochrome p450), GST (Glutathione S-transferase), GR (glucocorticoid
receptor), GSH (glutathione), PNPH (p-nitrophenol hydroxylase), TNF-α (tumor necrosis factor alpha),
IL-6 (interleukin-6).
10. Dosage and Bioavailability

The effects of RE have been studied in many cancer cell lines and although the concentrations
used in the in vitro studies are variable (0.1–500 µg/mL) it appears that the concentrations in the
range of 0.1–100 µg/mL are most effective. Similar to in vitro studies, the reported doses of RE used
in vivo are within a wide range (1 mg/mL drinking water −3333.3 mg/kg/day). This high variability
suggests the need for more systematic studies to identify effective RE doses in vivo. One study has
examined the levels of RE components in the plasma and tissue samples of animals administered
with RE. Administration of a single dose of RE (100 mg/mL water) enriched in CA (40% w/w) by
intragastric gavage in rats was followed by measurements of RE compounds and metabolites in plasma,
liver, small intestine content and brain. The researchers tentatively identified 26 compounds and the
main metabolites detected in plasma, liver and gut were glucuronide conjugates of CA, carnosol and
rosmanol [115]. Metabolites were detected as early as 25 min after oral administration and most of the
compounds remained present at substantial concentrations (micromolar range) for several hours [115].
Doolaege, et al. reported that 64.3 mg/kg (193.43 mM) CA orally administered to rats resulted in a
plasma concentration of 0.015 mg/mL (45.12 µM) [116]. Another study reported that ingestion of
360 mg/kg/day RA after 8 weeks resulted in a plasma concentration of 1.1 µM [117]. The reported
plasma concentrations of CA, carnosol and their metabolites were in the micromolar range indicating
that absorption and bioavailability are likely not barriers for these components of RE [114,115,118].
Another important issue that must be systematically examined in well-designed studies are the
potential toxicity of chronic administration of RE and RE polyphenols. Rosemary extract has already
been approved as a safe food additive by the European Food and Safety Authority (EFSA) [119] and
Nutrients 2016, 8, 731 24 of 32
is considered to be generally recognized as safe by the United States Food and Drug Administration
(FDA) (21CFR182.10). In a study reviewed by the EFSA, rosemary was found to have low acute and
sub-chronic toxicity in rats and the only effect at high doses was a slight increase in relative liver
weight, which has been shown to be reversible. Overall, 90 day RE administration (180–400 mg/kg/day,
equivalent to 20–60 mg/kg/day of carnosol plus CA) in rats revealed no observed adverse effect
levels (NOAEL) (reviewed in [119]). Furthermore, an acute single dose of 24 and 28.5 g/kg RE to
female and male mice respectively or the daily administration of 11.8 and 14.1 g/kg to female and
male mice respectively for 5 days resulted in no gross macroscopic lesions observed on autopsy
besides fatty liver in mice subjected to repeat administration of the extract indicating low acute toxicity
(reviewed in [119]). In another study, it was reported that an LD50 of 169.9 mg/kg/day RA was found
in mice implanted with Lewis lung carcinoma cells [90]. One study performed in humans used a
powdered RE mixed with citrus extract (1:1 ratio) (Nutroxsun™) which was consumed daily (250 mg)
for 3 months. Results showed a protective effect against UV-induced skin damage. Significant results
were seen after 8 weeks and continued to increase after 85 days of treatment [120]. Overall, the limited
in vivo studies report doses of RE or RE components that are relatively high and showed minimal
to no adverse effects, indicating low toxicity. Nonetheless, further research should be performed to
confirm maximum recommended doses of RE and RE components.
In humans, to achieve RE polyphenol levels that will provide health benefits high intake of
rosemary would be required, which is not practical. A more reasonable direction for the potential
future use of RE and its polyphenols as anticancer agents would be to develop easily ingestible and
soluble pills containing RE or RE components. Overall, the studies available currently suggest that
RE and its polyphenols CA and RA are good candidates for drug development and further research
examining the effective doses in animals is required before any clinical studies in humans are initiated.
In addition, systematic studies in animals to examine if chronic administration results in any toxicity
are required before clinical human studies.
It should be noted that in recent years, scientists have recognized that the gut microbiota plays an
important role in overall health and disease prevention. Although certain plant bioactive compounds
may be poorly bioavailable, the gut bacteria may generate metabolites that are more potent than the
parent compounds. A recent study found that administration of RE rich in CA (40% w/w) in rats had
a selective effect on caecum microbiota (increased the Blautia coccoides and Bacteroides/Prevotella
groups and reduced the Lactobacillus/Leuconostoc/Pediococccus group), decreased β-glucosidase
activity and increased fiber fecal elimination [121]. These data are associated with the decreased body
weight and the improvement of the metabolic and inflammatory status seen with RE [121]. Although
the above study suggests a potential prebiotic effect of RE administration against metabolic disorders
and obesity, there are no studies specifically examining the effect of gut microbiota on RE metabolites.
11. Conclusions
It should be noted that the levels of polyphenols and bioactive compounds present in RE may be
affected by many factors such as the plant growing conditions (soil, climate, exposure to stressors).
Additionally, the extraction method and storage of RE may affect its potency. Water, methanol, ethanol
and supercritical carbon dioxide extraction are methods which have been used in different studies and
evidence suggests that methanol (alcoholic-solvent) extraction may lead to RE with higher potency
(lower IC50) [31]. Since the source and extraction method of RE may affect its potency/biological
activity, this issue should be taken into consideration when future studies are planned.
In recent years, focus has shifted towards establishing new targeted cancer treatments that can
modulate specific pathways often mutated in cancer. RE and its polyphenols CA and RA may be
used as chemicals to target specific pathways leading to induction of apoptosis and decreased cell
survival. In addition, RE, CA and RA may be used as neutraceuticals to enhance the anticancer effects
of current chemotherapeutics. This could allow for lower doses to be used and less toxicity induced in
healthy surrounding tissue. Although studies examining signalling molecules and pathways targeted
Nutrients 2016, 8, 731 25 of 32
by RE, CA and RA are limited, the existing studies provide supporting evidence for the use of these
compounds both on their own and in combination with other cancer therapies.
Overall, RE, CA and RA have been shown to have various potent and effective anticancer
properties. However, more systematic studies are required in animals before human studies are
initiated. The in vivo animal studies should find (1) the doses to be administered; (2) the best route of
administration; (3) the plasma levels of CA, RA and other RE bioactive ingredients; (4) the signaling
molecules/pathways affected; and (5) any possible toxic effects associated with chronic administration.
Acknowledgments: This work was supported in part by a Brock University Advancement Fund (BUAF) grant to
E.T.
Author Contributions: J.M. and E.T. formulated the review topic, wrote the manuscript and reviewed the
manuscript. M.Y. contributed to writing and reviewing the manuscript. All authors read and approved the
final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

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nutrients

Received: 23 August 2016; Accepted: 8 November 2016; Published: 17 November 2016

Nutrients 2016, 8, 731; doi:10.3390/nu8110731 www.mdpi.com/journal/nutrients

2. Anticancer Effects of Rosemary Extract (RE): In Vitro Studies

Cancer Cell Dose/Duration Findings Mechanism Reference

Cancer Cell Dose/Duration Findings Mechanism Reference

Cancer Cell Dose/Duration Findings Mechanism Reference

Cancer Cell Dose/Duration Findings Mechanism Reference

Cancer Cell Dose/Duration Findings Mechanism Reference

Table 5. Anticancer effects of Rosemary Extract (RE). In vitro studies: leukemia.

Cancer Cell Dose/Duration Findings Mechanism Reference

3. Anticancer Effects of Rosemary Extract (RE): In Vivo Animal Studies

Table 6. Anticancer effects of Rosemary Extract (RE). In vivo studies.

Animal Model Dose/Duration Findings Mechanism Reference

4. Mechanisms of Anticancer Effects of Rosemary Extract (RE): In Vitro Studies

5. Mechanisms of Anticancer Effects of Rosemary Extract (RE): In Vivo Animal Studies

6. Anticancer Effects of Carnosic Acid (CA): In Vitro Studies

Cell Type Dose/Duration Findings Mechanism Reference

Cell Type Dose/Duration Findings Mechanism Reference

Cell Type Dose/Duration Findings Mechanism Reference

Cell Type Dose/Duration Findings Mechanism Reference

Cell Type Dose/Duration Findings Mechanism Reference

Table 11. Cont.

Cell Type Dose/Duration Findings Mechanism Reference

7. Anticancer Effects of Carnosic Acid (CA): In Vivo Animal Studies

Table 12. Anticancer effects of Carnosic Acid (CA). In vivo studies.

Animal Model Dose/Duration Findings Mechanism Reference

8. Anticancer Effects of Rosmarinic Acid (RA): In Vitro Studies

Cell Type Dose and Duration Findings Mechanisms Reference

Cell Type Dose and Duration Findings Mechanisms Reference

Cell Type Dose and Duration Findings Mechanisms Reference

Table 15. Cont.

Cell Type Dose and Duration Findings Mechanisms Reference

9. Anticancer Effects of Rosmarinic Acid (RA): In Vivo Animal Studies

Table 16. Anticancer effects of Rosmarinic Acid (RA). In vivo studies.

Animal Model Dose and Duration Findings Mechanisms Reference

Table 16. Cont.

Animal Model Dose and Duration Findings Mechanisms Reference

10. Dosage and Bioavailability

dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis. Afr. J. Tradit. Complement.

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