Pharmacological Properties of Protocatechuic Acid and Its Potential Roles as Complementary Medicine

This paper reviews the reported pharmacological properties of protocatechuic acid (PCA, 3,4-dihydroxy benzoic acid), a type of phenolic acid found in many food plants such as olives and white grapes. PCA is a major metabolite of anthocyanin. The pharmacological actions of PCA have been shown to include strong in vitro and in vivo antioxidant activity. In in vivo experiments using rats and mice, PCA has been shown to exert anti-inflammatory as well as antihyperglycemic and antiapoptotic activities. Furthermore, PCA has been shown to inhibit chemical carcinogenesis and exert proapoptotic and antiproliferative effects in different cancerous tissues. Moreover, in vitro studies have shown PCA to have antimicrobial activities and also to exert synergistic interaction with some antibiotics against resistant pathogens. This review aims to comprehensively summarize the pharmacological properties of PCA reported to date with an emphasis on its biological properties and mechanisms of action which could be therapeutically useful in a clinical setting.

Recently, several investigations have shown that PCA is a major metabolite of complex polyphenols, especially anthocyanins [4,5]. Anthocyanins have been shown to affect a variety of physiological activities which are of great benefit to health, including a reduced risk of cardiovascular diseases. This particular beneficial effect is partly due to the antiinflammatory properties [6][7][8], antioxidant and free radical scavenging activities [9][10][11][12], peroxidation inhibition [13], and estrogenic/antiestrogenic activity [14] of PCA. PCA is of particular nutritional interest since it is a main anthocyanin metabolite that can reach tissues in amounts which can exert biological effects on health [15]. In vivo studies demonstrated that male balb/cA mice which were fed a standard diet supplemented with PCA for 12 weeks showed increased PCA levels in plasma and tissues such as brain, heart, liver, and kidney [16]. Moreover, PCA itself has been shown to possess antioxidant properties as well as having other potential health benefits such as anti-inflammatory effects.
The aim of this review is to comprehensively summarize the pharmacological properties of PCA reported to date including antioxidant, anti-inflammatory, antihyperglycemia, antiapoptosis/proapoptosis, and antimicrobial activities, with an emphasis on the biological properties and mechanisms of action which could be potentially useful in a clinical setting.

Antioxidant Activity of PCA
Oxidative stress plays a key role in the pathogenesis of degenerative diseases such as cardiovascular diseases, diabetes mellitus, neurodegenerative diseases, cancer, and aging [17][18][19][20][21]. Mounting evidence from both in vitro and in vivo studies demonstrates that PCA exerts potent antioxidative effects. In in vitro studies, as summarized in Table 1, PCA was shown to have free radical scavenging and antioxidant activities by decreasing lipid peroxidation and increasing the scavenging of hydrogen peroxide (H 2 O 2 ) and diphenylpicrylhydrazyl (DPPH) [22]. In J77A.1 macrophage, PCA decreased oxidized low-density lipoprotein levels (LDL), inhibited superoxide (O 2 • ) and H 2 O 2 production, and also restored glutathione (GSH) related enzymes via c-Jun N-terminal kinase (JNK) mediated nuclear factor (erythroid-derived 2) like 2 (Nrf2) activation [23,24]. PCA also reduced reactive oxygen species (ROS) induced apoptosis by improving mitochondrial function, inhibiting DNA fragmentation in H 2 O 2 -induced oxidative stress in human neuronal cells [25], preventing lactate dehydrogenase (LDH) release in H 2 O 2 -induced oxidative stress in PC12 cells [26], and inhibiting intracellular ROS level in BNLCL2 cells [27].
Consistent with in vitro reports, in vivo studies (as summarized in Table 2) also demonstrated that PCA treatment decreased oxidative stress by promoting endogenous antioxidant enzymes in aging rats and also reduced H 2 O 2 -induced oxidative damage in aging mice, thus indicating that PCA could prevent oxidative damage in aging animals [26,28]. PCA also decreased advance glycation end products (AGEs) and ROS production in D-galactose-induced ROS and AGEs formation in mice [29]. In streptozotocin (STZ) induced diabetic rats, PCA was also found to decrease ROS formation in liver, heart, kidney, and brain by restoring endogenous antioxidant enzyme activities [3,30]. All of these findings indicated that the PCA possess potential antioxidant activity, suggesting that it could be used as a complementary medication to prevent oxidative damage in various degenerative diseases.

Anti-Inflammatory Activity of PCA
The inflammatory process is regulated by coordinated activation of both pro-and anti-inflammatory mediators in tissue cells (such as fibroblasts, endothelial cells, tissue macrophages, and mast cells) and also by the recruitment of leucocytes [31,32]. Prolonged activation of proinflammatory mediators causes tissue injury and organ dysfunction. As a consequence, chronic inflammation plays a critical role in the pathophysiology of major chronic diseases including obesity, cardiovascular disease, diabetes mellitus, Alzheimer's disease, and many types of cancer [33,34]. The mediators, including nitric oxide (NO), lipid mediators, cytokines/chemokines, adhesion molecules, and matrix metalloproteinases (MMPs), are involved in the initiation, maintenance, and resolution of the inflammatory process [35,36]. A summary of in vitro studies regarding the effects of PCA on the inflammatory process is shown in Table 3. PCA was shown to suppress tumor necrosis factor alpha (TNF-), interleukin-(IL-) 1 , inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2) expression via the regulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and mitogen-activated protein kinase (MAPK) activation in lipopolysaccharide-(LPS-) induced RAW 264.7 cell damage [37]. Moreover, PCA also suppressed vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) mRNA expression in TNF--induced cellular damage [38] and inhibited monocyte infiltration [39].
Consistent with in vitro reports, animal studies (Table 4) demonstrated that PCA strongly inhibited inflammation by inhibiting carrageenan-induced inflammation in mice by decreasing TNF-, IL-1 , and prostaglandin E2 (PGE 2 ) levels, suppressed iNOS and COX-2 expression in apolipoprotein E (ApoE) deficient mice [37], prevented LPS-induced sepsis in mice via decreased NO levels and suppressed IL-10 [40], reduced VCAM-1 and ICAM-1 [38], and inhibited monocyte/macrophage infiltration in mice [39]. Moreover, PCA also prevented coagulation and inflammation in STZinduced diabetic rats by inhibiting the plasma levels of the plasminogen activator inhibitor 1 (PAI-1), antithrombin III (AT-III), protein C, C-reactive protein (CRP), and von Willebrand factor (vWF) and reduced IL-6, TNF-, and monocyte chemoattractant protein-1 (MCP-1) levels in the heart and kidneys [3]. These findings suggest that the antiinflammatory effects of PCA might be beneficial in various chronic degenerative diseases in which the inflammatory process plays an important part in the pathogenesis.

Antihyperglycemic Activity of PCA
Maintenance of glucose homeostasis by strict hormonal control is of the utmost importance to human physiology 4 Evidence-Based Complementary and Alternative Medicine  Evidence-Based Complementary and Alternative Medicine 5    [41,42]. Failure of the control of glucose levels, with defects in both insulin action and insulin secretion, can result in a metabolic syndrome which is a multisymptom disorder of energy homeostasis [43]. It has been demonstrated that peroxisome proliferator-activated receptor gamma (PPAR ) is one of several targets of insulin activity, which regulates the expression and activity of key players in the maintenance of glucose transport machinery efficiency, such as glucose transporter (GLUT) 4 and adiponectin [44,45]. In in vitro studies, as summarized in Table 5, PCA has been shown to exert an insulin-like activity in oxidized LDL-induced insulin resistance in adipocytes via increased PPAR activation [45]. Similarly, in vivo studies (Table 6) also demonstrated that PCA decreased blood glucose levels in STZ-induced diabetes via restored carbohydrate metabolic enzyme activity, increased plasma insulin level, and normalized the activity of pancreatic islets [3,30,46]. These findings suggest that PCA provides antihyperglycemic effects in addition to its reported antioxidant and anti-inflammatory effects.

Antiapoptosis versus Proapoptotic Activity of PCA
Polyphenols have been shown to improve cell survival and protect against cytotoxicity by inhibiting apoptosis [18]. However, they can also induce apoptosis and prevent tumor growth [47,48]. These opposite effects are mainly due to its effects on the controlling of the cell redox state. Evidence from in vitro studies (Table 7) revealed that PCA has cell-protective effects via increased IkB degradation and subsequent NF-kB activation in TNF--induced cell death [49], attenuated changes of the mitochondrial membrane Evidence-Based Complementary and Alternative Medicine 7  [58] permeability, decreased oxidative stress damage and increased Bcl-2 levels in 1-methyl-4-phenylpyridinium-(MPP + -) induced apoptotic cell death [50], decreased caspase-3 activity in isolated neuronal stem cells (NSCs) [51], and reduced LDH leakage in H 2 O 2 -induced apoptosis [52].
In MPP + -induced cell death, PCA treatment resulted in a return to normal cellular morphology and normal mitochondria [53]. Moreover, PCA has been shown to have cellprotective effects via antioxidant and scavenging activities [54]. Unlike the cells described in Table 7, evidence from cancer cell studies (Table 8) demonstrated that PCA can induce apoptosis and prevent the growth of tumor cells via causing reduced Bcl-2 protein, increased Bax protein expression in human leukemia (HL-60) cells [55], via activated JNK/p38 MAPK pathways and Fas/FasL pathways, increased translocation of Bax, and reduced Bcl-2 levels in human gastric adenocarcinoma cells [56] and via induced JNK and p38 MAPK pathways in HepG2 hepatocellular carcinoma cells [57]. Moreover, PCA also demonstrated anticancer properties by causing apoptosis or suppressing invasion and metastasis in human breast, lung, liver, cervix and prostate cancer cells [58]. Consistently, an in vivo study (Table 9) also demonstrated that PCA inhibited N-nitrosomethylbenzylamine (NMBA) induced esophageal tumorigenesis by its inhibitory effects on genes associated with inflammation in rats [59].

Antimicrobial Activity of PCA
In vitro studies (Table 10) demonstrated that PCA has an antimicrobial effect against gram positive and negative bacteria and fungi [60,61]. PCA also prevented contamination of meat by Campylobacter and aerobes, by decreasing lipid oxidation [62]. PCA exerted its antibacterial effects due to its ability to inhibit bacterial growth and increase the synergistic effects of antibiotics hence reducing the possibility of resistance to drugs [63]. These antimicrobial activities of PCA have been proposed as promising applications in both health protection and food preservation in order to avoid food-borne illnesses [62,64].

Conclusion
Growing evidence suggests the significant biological potential of PCA through the modulation of cellular signals involved in the control of oxidative stress and inflammation. Moreover, its antiapoptotic effects in normal cells and proapoptotic effects in cancer cells suggest definite benefits as a potential chemotherapeutic agent. However, much evidence of such properties has been collected from cellular and animal studies, while clinical studies are still lacking. Future clinical studies are needed to warrant the clinical usefulness of the PCA.