**6. Physiological properties of oleocanthal and its putative health benefits**

Research conducted by Beauchamp and colleagues (Beauchamp et al., 2005) demonstrated that oleocanthal inhibits cyclooxygenase (COX) enzymes in a dose-dependent manner, mimicking the anti-inflammatory action exerted by ibuprofen. Cyclooxygenase 1 and 2 (COX 1 and COX 2) enzymes are responsible for the conversion of arachidonic acid to

interest is the spatial location of irritation produced by oleocanthal. Irritating, pungent compounds often aggravate all regions in the oral cavity rather than acting on one localized area (Peyrot des Gachons et al., 2011), which implies a sensory receptor specific to oleocanthal exists in the oropharyngeal region of the oral cavity. Recent investigations have indeed verified that the TRPA1 is the receptor linked to oleocanthal and the anatomical location of this receptor has been found in the oropharyngeal region of the oral cavity

Fig. 3. Oleocanthal irritation occurs solely in the oropharyngeal region (area shaded in

**6. Physiological properties of oleocanthal and its putative health benefits** 

Research conducted by Beauchamp and colleagues (Beauchamp et al., 2005) demonstrated that oleocanthal inhibits cyclooxygenase (COX) enzymes in a dose-dependent manner, mimicking the anti-inflammatory action exerted by ibuprofen. Cyclooxygenase 1 and 2 (COX 1 and COX 2) enzymes are responsible for the conversion of arachidonic acid to

However, further research is required to investigate this.

A large variability among subjects in the perceived irritation from oleocanthal has been noted (Cicerale et al., 2009a). Such individual variation in perception of oleocanthal may be related directly to the quantity of the TRPA1 receptor, as has been reported for other oral stimuli such as the bitter compounds: 6-n-propylthiouracil (PROP) and phenylthiocarbamide (PTC) (Hansen et al., 2006), which activate the TAS2R38 bitter receptor (Hayes et al., 2011). The important link between the perceptual aspects of oleocanthal and health benefits is the notion that variation in sensitivity to oleocanthal irritation may relate to potential differences in sensitivity to the anti-inflammatory action of this compound.

(Peyrot des Gachons et al., 2011).

black) of the oral cavity.

prostaglandins and thromboxane which are produced in response to inflammatory or toxic stimuli. COX 1 and COX 2 can be harmful to the body. In particular, COX 2 has been implicated in the pathogenesis of several cancers in both human and animal studies (Harris et al., 2003, Boland et al., 2004, Subbaramaiah et al., 2002, Ristimäki et al., 2002), and may also play a role in atherosclerosis (Chenevard et al., 2003). The novel findings presented by Beauchamp and colleagues (Beauchamp et al., 2005) demonstrate that oleocanthal not only mimics the mode of ibuprofen action, it exhibits increased potency (compared with ibuprofen) in inhibiting COX 1 and COX 2 enzymes at equimolar concentrations. For instance, oleocanthal (25 µM) inhibited 41-57% of COX activity in comparison to ibuprofen (25 µM) which inhibited only 13-18% of COX activity.

Moreover, Beauchamp and colleagues (Beauchamp et al., 2005) put forth the suggestion that chronic ingestion of small quantities of oleocanthal via EVOO consumption, may be responsible, in part, for the lowered prevalence of disease associated with the Mediterranean diet. Thus, if an olive oil consumer ingests around 50 g of EVOO a day containing approximately 200 μg/kg of oleocanthal, the person would ingest approximately 10 mg/day of oleocanthal. This would equate to a relatively low (10%) equivalent dose of ibuprofen (recommended for adult pain relief). Chronic low doses of ibuprofen and other COX inhibitors such as aspirin are known to have important health benefits in the prevention of cancer development (e.g. colon and breast) (Garcia-Rodriguez and Huerta-Alvarez, 2001, Harris et al., 2006) and CVD (Hennekens, 2002). Therefore, long term ingestion of oleocanthal via EVOO consumption may contribute to a reduction in chronic disease development and certainly emerging evidence supports this notion.

Finally, it is important to note oleocanthal's bioavailability within the body. Only one study has investigated this to date. A study by Garcia-Villalba et al. (Garcia-Villalba et al., 2010) noted that oleocanthal was readily metabolized however further studies are required to gain a more thorough understanding of the metabolism and bioavailability of this compound.

#### **6.1 Oleocanthal and neuro-degenerative disease**

Ibuprofen is known to exert beneficial effects on markers of neuro-degenerative disease (Van Dam et al., 2008) and based on the similar oral irritant properties and shared antiinflammatory mode of action via COX inhibition, oleocanthal was investigated for potential neuro-protective properties. Li and collegues (Li et al., 2009) presented significant findings demonstrating that oleocanthal inhibits tau fibrillization *in vitro* by forming an adduct with PHF6 peptide, which is a VQIXXK motif that resides in the microtubule binding region, and is crucial in the formation of tau fibrils (Li and Virginia, 2006) Hyperphosphorylated tangles of tau are lesions that are observed in neuro-degenerative disease (i.e. Alzheimer's disease) and PHF6 enables the phosphorylation of tau, thus the convalent modification of PHF6 peptide disrupts tau-tau interaction and subsequent fibril formation (Li et al., 2009) (Figure 4). Β-amyloid peptides (Aβ) are another type of lesion that are characteristic of Alzheimer's disease (Guela et al., 1998), as Aβ derived diffusible ligands (ADDLs) are neurotoxic factors proposed to instigate the onset of Alzheimer's disease (Pitt et al., 2009). Pitt and colleagues (2009) demonstrated that *in vitro*, oleocanthal alters the structure of ADDLs and augments antibody clearance of ADDLs, therefore protecting hippocampal neurons from ADDL toxicity. This data supports research showing a 40% decrease in Alzheimer's disease in populations consuming a Mediterranean style diet (Scarmeas et al., 2009).

Oleocanthal: A Naturally Occurring Anti-Inflammatory Agent in Virgin Olive Oil 363

Fig. 5. Nitric oxide (NO) derived from nitric oxide synthase (NOS), functions as a neurotransmitter and vasodilator, and is important in normal physiological responses. Inducible nitric oxide synthase (iNOS) is a third form of NOS and is not present in resting cells, but rather is induced by inflammatory cytokines. NO produced from iNOS promotes inflammation in chrondocytes and is associated with cartilage degenerative diseases such as

research to consolidate the pharmacological potential of this compound.

Recent evidence demonstrates that oleocanthal may exert therapeutic properties against the pathogenesis of c-Met kinase induced malignancies. Elnagar and colleagues (Elnagar et al., 2011) have reported that oleocanthal possesses anti-proliferative effects in human breast and prostate cancer lines (Elnagar et al., 2011). Also Khanal and collegues (Khanal et al., 2011) recently showed that oleocanthal has an anti-proliferative effect and prevents tumour induced cell transformation in mouse epidermal JB6 Cl41cells. The mechanism of action in which oleocanthal achieved this was via the inhibition of extracellaullar signal-regulated kinases 1/2 and p90RSK phosphorylation. Furthermore, oleocanthal has also been shown to promote cell apoptosis by activating caspase-3 and poly-adenosine diphosphate-ribose polymerase, the phosphorylation of p53 (Ser15), and also induced fragmentation of DNA in HT-29 cells derived from human colon adenocarcinoma (Khanal et al., 2011). These findings suggest that oleocanthal may have potential as a therapeutic agent in the inhibition of carcinoma progression and supports substantial evidence that populations residing in the Mediterranean region have a reduced incidence of prostate, breast, lung and gastrointestinal cancer (Trichopoulou et al., 2000, La Vecchia, 2004, Fortes et al., 2003, Dixon et al., 2007). It is important to note that while there is strong evidence that oleocanthal is an effective antiinflammatory agent and demonstrates pharmacological characteristics *in vitro,* future *in vivo*  studies are required to fully elucidate the efficacy of this natural NSAID. Caution is required when extrapolating results from a single compound out of the matrix in which it normally exists. Oleocanthal is one of many phenolic compounds contained in EVOO, and it is probable that the synergistic and interactive actions of these phenolics combined are responsible for the low incidence of chronic inflammation associated with EVOO intake. Furthermore, the bioavailability of oleocanthal needs to be firmly established in future

osteoarthritis.

**6.3 Oleocanthal and cancer** 

Fig. 4. Oleocanthal inhibits tau fibrilisation by covalently modifying the PHF-6 peptide which is crucial for the formation of tau fibrils. The fibrilisation of tau leads to neurofibrillary tangles which are inherently associated with neurodegenerative diseases such as Alzheimer's disease.

#### **6.2 Oleocanthal and joint-degenerative disease**

*In vitro* research draws attention to oleocanthal, as a potential therapeutic compound that may be of interest in the quest to find suitable natural NSAIDs for the treatment of joint degenerative disease. Pro-inflammatory cytokines up-regulate the synthesis of cartilage to degrading enzymes, and stimulate nitric oxide (NO) production (Scher et al., 2007), as well as increase prostaglandin PGE2 production, which have all been implicated in the development of arthritic pain and thus joint-degenerative disease. COX enzymes are a catalyst for the formation of prostaglandins and have also been reported to be highly expressed in arthritic spine in an animal model (Procházková et al., 2009). Therefore, oleocanthal may influence arthritic pain through inhibition of PGE2 synthesis accompanying COX inhibition.

NO plays an integral role in joint-degenerative disease and the stable end product of NO, nitrite (NO2), is significantly expressed in arthritic synovial fluid (Iacono et al., 2010). In osteoarthritis arthritis (OA) pathogenesis, diseased cartilage synthesizes NO spontaneously from diseased chrondocytes (Tung et al., 2002). NO is biosynthesized by nitric oxide synthase (NOS). Another form of NOS is inducible NOS (iNOS) which is largely responsible for the inflammatory actions of NOS (Espey et al., 2000) (Figure 5). Iacano and collegues (2010) have shown that oleocanthal and synthesized derivatives, decrease production of iNOS protein expression in LPS challenged murine chondrocytes, dose dependently, further highlighting the anti-inflammatory actions of oleocanthal and the pharmacological potential. Also, as oleocanthal mediates prostaglandin synthesis via inhibitory actions on COX enzymes, it is possible that oleocanthal may exert pharmacological actions in the treatment of both rheumatoid arthritis and osteoarthritis through COX inhibition.

Fig. 5. Nitric oxide (NO) derived from nitric oxide synthase (NOS), functions as a neurotransmitter and vasodilator, and is important in normal physiological responses. Inducible nitric oxide synthase (iNOS) is a third form of NOS and is not present in resting cells, but rather is induced by inflammatory cytokines. NO produced from iNOS promotes inflammation in chrondocytes and is associated with cartilage degenerative diseases such as osteoarthritis.

#### **6.3 Oleocanthal and cancer**

362 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

Fig. 4. Oleocanthal inhibits tau fibrilisation by covalently modifying the PHF-6 peptide which is crucial for the formation of tau fibrils. The fibrilisation of tau leads to neurofibrillary tangles which are inherently associated with neurodegenerative diseases such as Alzheimer's disease.

*In vitro* research draws attention to oleocanthal, as a potential therapeutic compound that may be of interest in the quest to find suitable natural NSAIDs for the treatment of joint degenerative disease. Pro-inflammatory cytokines up-regulate the synthesis of cartilage to degrading enzymes, and stimulate nitric oxide (NO) production (Scher et al., 2007), as well as increase prostaglandin PGE2 production, which have all been implicated in the development of arthritic pain and thus joint-degenerative disease. COX enzymes are a catalyst for the formation of prostaglandins and have also been reported to be highly expressed in arthritic spine in an animal model (Procházková et al., 2009). Therefore, oleocanthal may influence

NO plays an integral role in joint-degenerative disease and the stable end product of NO, nitrite (NO2), is significantly expressed in arthritic synovial fluid (Iacono et al., 2010). In osteoarthritis arthritis (OA) pathogenesis, diseased cartilage synthesizes NO spontaneously from diseased chrondocytes (Tung et al., 2002). NO is biosynthesized by nitric oxide synthase (NOS). Another form of NOS is inducible NOS (iNOS) which is largely responsible for the inflammatory actions of NOS (Espey et al., 2000) (Figure 5). Iacano and collegues (2010) have shown that oleocanthal and synthesized derivatives, decrease production of iNOS protein expression in LPS challenged murine chondrocytes, dose dependently, further highlighting the anti-inflammatory actions of oleocanthal and the pharmacological potential. Also, as oleocanthal mediates prostaglandin synthesis via inhibitory actions on COX enzymes, it is possible that oleocanthal may exert pharmacological actions in the

arthritic pain through inhibition of PGE2 synthesis accompanying COX inhibition.

treatment of both rheumatoid arthritis and osteoarthritis through COX inhibition.

**6.2 Oleocanthal and joint-degenerative disease** 

Recent evidence demonstrates that oleocanthal may exert therapeutic properties against the pathogenesis of c-Met kinase induced malignancies. Elnagar and colleagues (Elnagar et al., 2011) have reported that oleocanthal possesses anti-proliferative effects in human breast and prostate cancer lines (Elnagar et al., 2011). Also Khanal and collegues (Khanal et al., 2011) recently showed that oleocanthal has an anti-proliferative effect and prevents tumour induced cell transformation in mouse epidermal JB6 Cl41cells. The mechanism of action in which oleocanthal achieved this was via the inhibition of extracellaullar signal-regulated kinases 1/2 and p90RSK phosphorylation. Furthermore, oleocanthal has also been shown to promote cell apoptosis by activating caspase-3 and poly-adenosine diphosphate-ribose polymerase, the phosphorylation of p53 (Ser15), and also induced fragmentation of DNA in HT-29 cells derived from human colon adenocarcinoma (Khanal et al., 2011). These findings suggest that oleocanthal may have potential as a therapeutic agent in the inhibition of carcinoma progression and supports substantial evidence that populations residing in the Mediterranean region have a reduced incidence of prostate, breast, lung and gastrointestinal cancer (Trichopoulou et al., 2000, La Vecchia, 2004, Fortes et al., 2003, Dixon et al., 2007). It is important to note that while there is strong evidence that oleocanthal is an effective antiinflammatory agent and demonstrates pharmacological characteristics *in vitro,* future *in vivo*  studies are required to fully elucidate the efficacy of this natural NSAID. Caution is required when extrapolating results from a single compound out of the matrix in which it normally exists. Oleocanthal is one of many phenolic compounds contained in EVOO, and it is probable that the synergistic and interactive actions of these phenolics combined are responsible for the low incidence of chronic inflammation associated with EVOO intake. Furthermore, the bioavailability of oleocanthal needs to be firmly established in future research to consolidate the pharmacological potential of this compound.

Oleocanthal: A Naturally Occurring Anti-Inflammatory Agent in Virgin Olive Oil 365

Time Gradient

Table 1. Mobile phase gradient for oleocanthal separation.

0-35 min 75% water, 25% acetonitrile 36-55 min 90% acetonitrile, 10% methanol 56-65 min 75% water, 25% acetonitrile

Fig. 7. Negative ion mass spectrum of oleocanthal with the characteristic [M-H]-

the olives to oil, storage of oil, and domestic heat application to oil.

**8. Oleocanthal concentration in olive oil and factors which may affect its** 

Oleocanthal concentration in EVOO is highly variable, ranging from as little as 0.2 mg/kg to as high as 498 mg/kg (Gomez-Rico et al., 2006). Such variation in oleocanthal concentration amongst differing EVOOs may be due to multiple factors that have the capacity to modify the concentration of this compound (Carrasco-Pancorbo et al., 2005). These factors include: method of phenolic extraction and quantification, geographic region of olive growth, olive tree cultivar, agricultural techniques applied to cultivate olives, olive maturity, processing of

The analytical method used to quantify phenolics present in EVOO has an influence on the reported concentration and therefore is an important consideration when interpreting and comparing the data of such investigations (Carrasco-Pancorbo et al., 2005). Regarding the

303.12 (Cicerale et al., 2009b).

**8.1 Extraction and quantification** 

**concentration** 

ion at *m/z*
