**1. Introduction**

Rosmarinic acid (RA) is an ester of caffeic acid and 3,4-dihydroxyphenyl lactic acid that occurs in nature as phenolic compounds. Its molecular formula is C18H16O8 and is formally known as (R)-α-[[3-(3,4-dihydroxyphenyl)-1-oxo-2 E-propenyl]oxy]-3,4-dihydroxy-enzenepropanoic acid (Figure 1). The main sources of RA are plants belonging to the Boraginaceae family, subfamily Nepetoideae. It was isolated for the first time in 1958 from the rosemary plant (*Rosmarinus o*ffi*cinalis* L.), and recently it has been reported in *Forsythia koreana* (Rehder) Nakai, *Hyptis pectinate* (L.) Poit., *Ocimum tenuiflorum* L., *Thymus mastichina* (L.) L., and plants belonging to Lamiaceae family [1].

**Figure 1.** Chemical structure of rosmarinic acid.

RA has remarkable biological effects, including antiviral, antibacterial, anticancer, antioxidant, anti-aging, antidiabetic, cardioprotective, hepatoprotective, nephroprotective, antidepressant, antiallergic, and anti-inflammatory activities (Figure 2). RA and some rosemary extract-isolated compounds, like carnosic and ursolic acids and carnosol have also shown to be able to reduce the likelihood of tumor development in several body organs, such as stomach, colon, liver, breast, and leukemia cells [2–4]. Thus, here we review the various therapeutic potentials of RA in this article.

#### **2. Bioavailability of Rosmarinic Acid and Its Metabolic Changes in the Human Body**

RA is partially metabolized to the coumaric acid and caffeic acid in the body of the rat [5], and the hypolipidemic effect of RA may also be a consequence of the action of its metabolites. For example, caffeic acid inhibited the synthesis of hepatic fatty acid synthase, 3-hydroxy-3-methylglutaryl CoA reductase and acyl-CoA:cholesterol acyltransferase activities and increased fatty acid β-oxidation activity in high-fat diet-induced obese mice [6]. Caffeic acid and sinapic acid increased serum estradiol concentrations in rats with estrogen deficiency, which may have contributed to the observed metabolic effects [7]. In the rat ovary ovulation, external ovarian tissues such as fatty tissue, skin, bones and brain are the source of estradiol. In these sites, C19 cannot be synthetized. Steroids C19 (androgens) can be converted to estrogens by aromatase. Therefore, it seems possible that RA or its metabolites increase the activity of aromatase. Caffeic acid increased estradiol and reduced total cholesterol concentrations only in rats that were fed standard food containing soy, and these effects were not observed in rats fed without soy with reduced phenolic acid contents [8]. It is therefore possible that at least some of the RA effects reported depend on the diet. RA showed similar beneficial effects on some lipid parameters and insulin resistance (HOMA-IR) as that demonstrated for sinapic acid in a parallel study [7]. Moreover, RA had positive effects on expression of hepatic genes or proteins involved in signaling insulin and glucose and lipid metabolism, such as insulin receptor substrate-1 (IRS-1), 5 AMP-activated protein kinase (AMPK), phosphoenolpyruvate carboxykinase (PEPCK), glucose transporter 2 (GLUT2), forkhead box protein O1 (FOXO1), sterol regulatory element-binding protein 1 (SREBP1), and carnitine palmitoyltransferase 1 (CPT1) in diabetic rats [9]. The possible mechanism of action of RA on glucose and lipid metabolism may be mediated by peroxisome proliferator-activated receptor (PPAR) peroxidation; RA has been shown to activate these receptors. It should be noted that the lower RA dose (10 mg/kg) was sufficient to reduce the HOMA-IR index and the concentration of fructosamine, while a higher dose (50 mg/kg) was required to reduce the total cholesterol and

triglyceride levels in rats with estrogen deficiency. Moreover, RA and its metabolites can directly neutralize reactive oxygen species (ROS) [10] and thereby reduce the formation of oxidative damage products. The antioxidant activity of RA directly derives from its structure, namely the presence of 4 hydrogens in the phenolic system and two catecholic moieties, which give this compound polar character. Electrochemical studies have shown that RA oxidizes in two steps. In the first step, the rest of the caffeic acid is oxidized and in the second step the residue of 3,4-dihydroxyphenyl lactic acid.

**Figure 2.** Rosmarinic acid and its potential functions.

RA is therefore considered to be the strongest antioxidant of all hydroxycinnamic acid derivatives [11]. Inhibition of the production of advanced glycation end products under the influence of RA was previously presented in vitro and in vivo [12]. The use of RA in doses of 10 and 50 mg/kg in rats with estrogen deficiency did not affect the body mass. RA administered at a dose of 10 mg/kg of ovariectomized rats did not affect estradiol and progesterone concentrations compared with ovariectomy control rats, whereas RA at a dose of 50 mg/kg of estradiol showed a trend of growth. Orchids containing RA are often used in self-healing and daily diets, so it is possible to consume 5–10 g of these plants daily in the form of infusions and spices [13]. RA is water-soluble, and according to literature data, the efficacy of secretion of this compound in infusions is about 90% [14]. Accordingly, it is possible to consume approximately 110 mg RA daily, i.e., approximately 1.6 mg/kg for adult men weighing 70 kg. Increasing the concentration of reduced glutathione (GSH) in plasma due to the use of RA was previously described in various models of diabetes [15]. RA has been shown to stimulate the regulation of the catalytic subunits of the glutamate cysteine ligase (the enzyme involved

in the biosynthesis of GSH) in the hematopoietic stem cells [16]. It can be assumed that the increase in the concentration of GSH, after the administration of RA, was previously the result of the intense biosynthesis of GSH rather than its recovery from the oxidized form. Moreover, it should be noted that the RA appears to be absorbed into the rat mainly as its metabolites [5]. It is possible that these metabolites also play a role in the observed increase in GSH concentration. Furthermore, serum GSH/oxidized glutathione (GSSG) was calculated, as it is known to be an important indicator of redox cell status as well as for the state of redox at the tissue and whole body [17]. The adventitious e ffect of RA on redox homeostasis has been shown to increase the ratio of GSH/GSSG in serum rats.

#### **3. Health Benefits of Rosmarinic Acid**

#### *3.1. Anticancer Potential*

Several mechanisms have been proposed for RA anticancer activity (Figure 3). For instance, in rats with colon cancer, RA at the concentration of 5 mg/kg body weight (b.w.) impaired tumor formation and development, reduced lipid peroxidation by-products and pro-apoptotic proteins expression, modulated xenobiotic enzymes, and increased apoptotic proteins expression [18]. In human liver cancer cell line, HepG2, transfected with plasmid containing ARE-luciferin gene, RA predominantly enhances ARE-luciferin activity and promotes nuclear factor E2-related factor-2 (Nrf2) translocation from cytoplasm to the nucleus and also increases MRP2 and P-gp e fflux activity along with intercellular ATP level [19]. A study conducted by Wu et al. [20] reported that RA inhibited CCRF-CEM and CEM/ADR5000 cells in a dose-dependent pattern but caused less cytotoxicity towards normal lymphocytes. RA concurrently induced necrosis and apoptosis and stimulated MMP dysfunction activated PARP-cleavage and caspase-independent apoptosis. RA also blocked the translocation of p65 from the cytosol to the nucleus [20]. Moreover, it inhibits transcription factor hypoxia-inducible factor-1 α (HIF-1 α) expression, which a ffects the glycolytic pathway; meanwhile, it also suppressed glucose consumption and lactate production in colorectal cells [21]. RA also inhibits micro RNAs and pro-inflammatory cytokines and thus may suppress the Warburg e ffects through an inflammatory pathway involving activator of transcription-3 (STAT3) and signal transducer of interleukin (IL)-6 [22]. Furthermore, RA inhibits HL-60 promyelocytic leukemia cells' growth and development and provides strong scavenging free radical e ffects, disturbing the balance of nuclear deoxyribonucleotide triphosphate (dNTP) levels without a ffecting protein levels of RR (R1, R2, p53R2) subunits, ultimately leading to apoptosis induction [23,24].

RA application, at a concentration of 5 mg/kg b.w. during 30 weeks in 1,2-dimethyldrazin stimulated colon carcinogenesis in the rat at 20 mg/kg b.w. and significantly stopped tumor formation and proliferation. RA supplementation also reduced tumor necrosis factorα (TNFα), cyclooxygenase-2 (COX-2) and IL-6 levels, and modulated p65 expression [25]. It is also able to inhibit the release of the highly mobile group box 1 (HMGB1) and to slow down HMGB1-dependent inflammatory responses in human endothelial cells, stopping HMBG1-mediated hyperpermeability and leukocytes migration in mice [26]. RA supplementation primarily decreases aberrant cryp<sup>t</sup> foci (ACF) formation and multiplicity in rats [27].

RA inhibited APC10.1 cell growth that comes in Apc (Min) mouse model of colorectal carcinogenesis [28]. Through oral administration, RA totally prevented skin tumor cells formation in DMBA-induced mouse skin carcinogenesis and decreased lipid peroxidation byproducts levels [29]. It also inhibited human ovarian cancer A2780 cell line, disturbing the cell cycle at multiple phases and stimulating apoptosis by modifying multiple genes expression, involved in apoptosis regulation [30].

RA induced the cell cycle arrest and apoptosis in prostate cancer cell lines (PCa, PC-3, and DU145) [31]. These e ffects were mediated through modulation of histone deacetylases expression (HDACs), specifically HDAC2; the aberrant expression of theses enzymes is related with the onset of human cancer.

In rats with 1,2 dimethylhydrazine-induced colon carcinogenesis, RA administration at dose of 2.5, 5, and 10 mg/kg b.w. led to a decrease in the number of polyps (50%), reversed oxidative markers (21%), antioxidant status (38.6%), CYP450 contents (29.4%), and PNPH activities (21.9%) [32]. RA can also inhibit adhesion, invasion, and migration of Ls 174-T human colon carcinoma cells through enhancing GSH levels and decreasing ROS levels. Finally, RA may also inhibit colorectal carcinoma metastasis, by reducing the extracellular signal-regulated kinase pathway and the number and weight of lung tumors [33]. MDA-MB-231BO human bone homing breast cancer cells migrations are also inhibited by RA, whereas the number and size of mineralized nodules in ST-2 murine bone marrow stoma cells cultures raise [34].

RA also enhances chemosensitivity of human resistant gastric carcinoma SGC7901 cells [35]. The anticancer potential of RA analogues has also been tested. RA analogue-11 induces apoptosis of SGC7901 via the epidermal growth factor receptor (EGFR)/Akt/nuclear factor kappa B (NF-κB) pathway [36].

**Figure 3.** Mechanism of rosmarinic acid as an anticancer agent.

## *3.2. Antidiabetic Activity*

RA supplementation increases the expression of mitochondrial biogenesis key genes, like sirtuin 1 (SIRT-1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and mitochondrial transcription factor A (TFAM) via activation of AMP-activated protein kinase (AMPK) in the skeletal muscle of insulin-resistant rats as well as in L6 myotubes. It also increased glucose faster and decreased serine IRS-1 phosphorylation, while increasing the glucose transporter type 4 (GLUT4) transfer [37].

In streptozotocin (STZ)-induced diabetic rats, RA exerted a noticeable hypoglycemic effect, whereas in high-fat diet (HFD) fed diabetic rats it increased glucose utilization and ameliorated insulin sensitivity. RA supplementation inverted the STZ- and HFD-induced increase in phosphoenolpyruvate carboxykinase (PEPCK) expression in the liver and the STZ- and HFD-induced decrease in GLUT4 expression in skeletal muscle. RA exerts hypoglycemic effects and improves insulin sensitivity, also increasing GLUT4 expression and decreasing PEPCK expression [38]. In addition, it also reverses memory and learning defects through improving cognition in healthy rats, inhibiting hyperglycemia, lipid peroxidation, and enhancing antioxidant defense system [39]. At a concentration of 10 mg/kg, RA decreased TBARS levels in kidney and liver of STZ-induced diabetic rats. This effect was mainly conferred by its ability to increase superoxide dismutase (SOD) and catalase (CAT) activity and to reverse the decrease in ascorbic acid and of non-protein thiol levels in diabetic rats [40]. RA administration also ameliorated oxidative stress markers in diabetic rats and water consumption and urination. Thus, it was proposed that RA mitigates STZ-induced diabetic manifestations by protecting rat's tissues against free radicals' damaging e ffects [15]. At 100 mg/kg, RA significantly increased insulin index sensitivity and reduced blood glucose, advanced glycation end-products, HbA1c, IL-1β, TNF α, IL-6, p-JNK, P38 mitogen-activated protein kinase (MAPK), and NF-κB levels. Moreover, it significantly reduced free fatty acids (FFA), triglycerides, serum cholesterol, AOPPs, lipid peroxides, and protein carbonyls levels in plasma and pancreas of diabetic rats. The reduced activities of CAT, SOD, glutathione *S*-transferases (GST), and glutathione peroxidase (GPx) and the reduced levels of vitamins C and E, ceruloplasmin, and GSH in plasma of diabetic rats were also significantly recovered by RA application. Furthermore, it protects pancreatic β-cells from oxidative stress in HFD-STZ-induced experimental diabetes [41]. The protective e ffects of RA (30 mg/kg) against hypoglycemia, hyperlipidemia, oxidative stress, and an imbalanced gu<sup>t</sup> microbiota architecture was studied in diabetic rats. The treatment decreased the levels of fasting plasma glucose, total cholesterol, and triglyceride, exhibited an antioxidant and antiglycative effect, showed protective e ffects against tissue damage and inflammation in the abdominal aorta, increased the population of diabetes-resistant bacteria, and decreased the number of diabetes-sensitive bacteria [12].

RA also reduced diabetes occurrence and preserved normal insulin secretion, ROS, and reactive nitrogen species (RNS) by regulating antioxidant enzymes, and attenuating the pro-inflammatory T helper 2 and T regulatory cells levels [42]. At a concentration of 10 mg/kg, RA treatment significantly reduced lipid peroxidation levels in the hippocampus (28%), cortex (38%), and striatum (47%) of diabetic rats [43]. In Wistar rats, RA administrated orally at 50 mg/kg for 10 weeks in STZ-induced diabetes diminished endothelium-dependent relaxation accompanied by IL-1β, TNFα, preproendothelin-1, and endothelin converting enzyme 1 overexpression. It also provided aortic endothelial function protection against diabetes-induced damage [44]. Finally, it significantly inhibited the carbohydrate-induced adaptive increase of sodium-dependent glucose cotransporter 1 (SGLT1) in the enterocyte brush border membrane [45].
