The unsaponifiable fraction of olive oil corresponds to 1% to 2% of this food. It is composed of a large variety of compounds (Table 1). They are largely responsible for the taste and aroma (flavor) of olive oil, besides contributing to relevant biological activities. Interestingly, they can modulate the redox system and different cellular signaling pathways. Therefore, olive oil consumption may have effects on metabolism, in general, and physiological processes in MSCs, in particular, as described below and summarized in Figure 3.
Phenolic compounds of extra virgin olive oil represent 18% to 37% (100-300 mg/kg) of the unsaponifiable fraction. They are partially responsible for the high oxidative stability of olive oil. Their composition and concentration depend on the olive tree cultivar, weather conditions, fruit ripeness, and technology used for olive oil extraction. The main types of phenolic compounds of extra virgin olive oil are flavonoids, lignans, phenolic acids, phenolic alcohols and secoiridoids, the last two being the most abundant. Indeed, secoiridoids represent between 85% and 99% of the total phenolic compounds of olive oil. Their intake is healthy, being antioxidants (preventing oxidative stress) and interacting with several signaling pathways. Actually, they have antiinflammatory, anticarcinogenic, antiatherogenic and antithrombotic effects. Besides, they regulate lipidic metabolism, among other beneficial activities. Interestingly, the biological activity of phenolic compounds also modulates MSC physiology, including their proliferation, viability, regenerative capacity and differentiation, as demonstrated by numerous studies in the last two decades.
Oleuropein is a relevant secoiridoid, ranging from 1.2-120 mg/kg in extra virgin olive oil. It has numerous beneficial health properties, including antioxidant, antiinflammatory, antiatherogenic, anticancerogenic, antimicrobial, antiviral and antiaging effects, among others. Interestingly, oleuropein prevents both oxidative stress and apoptosis induced by H2O2 in MSCs. That is accomplished by inhibiting the B-cell lymphoma 2 (referred to as Bcl-2)-like protein 4 (referred to as BAX) proapoptotic protein, activating the antiapoptotic Bcl-2 and myeloid leukemia cell differentiation protein (referred to as Mcl-1), and modulating autophagy-related death signals.
Additionally, oleuropein also affects MSC differentiation. We have described that this phenolic compound upregulates osteoblastic marker genes, increasing alkaline phosphatase activity, mineralization and osteoprotegerin (OPG)/RANKL expression ratio in human MSCs derived from bone marrow. These results suggest that oleuropein favors osteogenesis, inhibiting bone resorption. The latter is accomplished by activating expression of OPG, which is a decoy receptor for the receptor activator of RANKL, which is an osteoclastogenesis activator. Therefore, an increase in OPG expression reduces the ability of RANKL to activate osteoclastogenesis.
We have reported that oleuropein inhibits adipogenic differentiation of MSCs by downregulating PPARγ and other adipogenic genes, reducing fat droplet formation in treated cultures. We have also carried out transcriptomic analyses of MSCs induced to differentiate into adipocytes in the presence of oleuropein. Interestingly, this compound restored expression of 60% of the genes repressed during adipogenesis, activating some signaling pathways, like Rho [family of guanosine triphosphate (GTP) ases] and beta-catenin, and inhibiting others related to mitochondrial activity, which are induced during adipogenesis. This indicates that the presence of oleuropein keeps MSCs that have been induced to adipocytes in a more undifferentiated state. Therefore, since this compound favors osteoblastogenesis versus adipogenesis, it can have osteoprotective properties and its consumption may prevent some diseases, like osteoporosis. It can also be beneficial in physiological processes affecting bone health, like aging. That rationale is supported by the fact that treatment with 10 mg of oleuropein/kg every 3 d prevented loss of trabecular bone in the femurs of ovariectomized mice. Besides, the authors also showed in in vitro studies that this compound favored mineralization of MC3T3-E1 preosteoblastic cells from mice and inhibited osteoclastogenesis.
Additionally, oleuropein can prevent formation of ectopic fat, preventing obesity. Indeed, it has been reported that this phenolic compound prevented formation of visceral fat in obese mice and inhibited adipogenesis in 3T3-L1 preadipocytes. Oleuropein may also have antiaging effects on MSCs, maintaining a better regenerative capacity of the organism with advanced age. This is because it can inhibit the phosphatidylinositol 3‐kinase/Akt/mammalian target of rapamycin (commonly known as mTOR) signaling pathway on MSCs. The inhibition of phosphoinositide 3-kinases (commonly known as PI3Ks)/Akt/mTOR pathway maintained high proliferative and differentiation capacities in MSCs. Akt stands for Ras-related C3 botulinum toxin substrate 1, (RAC)-alpha serine/threonine-protein kinase, also known as protein kinase B (or its more common abbreviation of PKB).
Hydroxytyrosol is another of the most abundant phenolic compounds present in extra virgin olive oil. Its concentration ranges from 1.1 mg/kg to 75 mg/kg. This phenolic alcohol has many healthy effects, including antiinflammatory, antimicrobial, cardioprotective, neuroprotective and antitumoral activities, among others. Thus, it has also been considered as a nutraceutical. Regarding its effect on MSCs, we have evaluated the effects of 1 μmol/L and 100 μmol/L hydroxytyrosol on osteogenic and adipogenic differentiation of MSCs from human bone marrow. The highest concentration of this compound (but not the lower one) reduced the number of cells, repressed expression of osteoblastic gene collagen type-I alpha-1 (COLIA1) and reduced mineralization in cultures induced to differentiate into osteoblasts. On the other hand, mostly the highest concentration increased both expression of the PPARγ gene as well as of generation of fat droplets, indicating induction of adipogenesis.
Yet, other authors have described that such high concentration of hydroxytyrosol inhibited adipogenesis in 3T3-L1 preadipocytes from mice[104,105]. Discrepancies were probably due to different cellular types and different methodologies used to induce differentiation. Interestingly, mitotic clonal expansion is an important event during adipogenic differentiation of 3T3-L1, but does not take place in human MSCs. This compound inhibited mitotic clonal expansion of 3T3-L1[104,105]. Therefore, its effects on mice versus human adipogenesis may be different. Additionally, it has been recently reported that 30 μg of hydroxytyrosol/mL (~ 200 μmol/L) inhibited adipogenesis on human preadipocytes (not affecting mature adipocytes), further increasing apoptosis.
Nevertheless, it should be taken into account that plasma levels of hydroxytyrosol have been found to be about 4.5 ng/mL (~ 0.029 μmol/L) in 30 min after consumption. Therefore, the effect of high concentrations of hydroxytyrosol, reported by others and ourselves, on osteoblastic and adipogenic differentiation of human MSCs in vitro would require a pharmacological in vivo intake. Interestingly, studies with animals have shown that both oleuropein and hydroxytyrosol protected 6-wk-old ovariectomized mice of trabecular (albeit not cortical) bone loss in femur. Yet, it is not clear if that was due to increased osteoblastogenesis, inhibition of osteoclastogenesis, or both.
Additionally, hydroxytyrosol has positive effects on chondrocytes, which are other cells also derived from MSCs. For instance, in an osteoarthritis model in which chondrocytes have been stimulated with growth-related oncogene alpha (commonly known as GROα) to promote hypertrophy and terminal differentiation, the presence of hydroxytyrosol reduced oxidative stress and apoptosis, which are induced in such pathology. These results suggest that this compound might have a relevant role in MSC physiology in bone marrow, modulating bone metabolism. Nevertheless, further research is required to properly ascertain its putative effects in vivo.
Extra virgin olive oil also contains other phenols, known as flavonoids, mainly luteolin and apigenin. Their concentrations range from 0-19 mg/kg, therefore representing a minority. Despite that, their intake has been associated with reduced risk to suffer cardiovascular disease, cancer, and neurodegenerative disorders. Both flavonoids upregulate the octamer-binding protein 4 (OCT4) and sex-determining region Y box-containing gene 2 (SOX2) genes in MSCs. Such genes are mainly expressed in embryonic stem cells, encoding transcription factors that regulate cell cycle and maintenance of totipotency or pluripotency. They are considered additional MSC molecular markers in adult tissues, being repressed after cell differentiation. Therefore, luteolin and apigenin may delay the loss of regenerative capacity of MSCs with aging. In addition, also related to aging, luteolin prevented oxidative stress in vitro, induced with FeCl2 and H2O2 in MSCs.
Luteolin can also affect MSC differentiation. Indeed, it inhibited adipogenic differentiation of 3T3-L1 murine cells, reducing triglyceride accumulation, as well as downregulating genes encoding the PPARγ and CCAAT-enhancer-binding proteins (C/EBPα) adipogenic transcription-factors. Regarding osteogenic differentiation, some authors have reported that luteolin reduced alkaline phosphatase activity and viability of MC3T3-E1 preosteoblastic cells. Yet, other authors using the same cellular type but much lower concentrations of this flavonoid have found that it prevented the osteoblastogenesis inhibition induced by glucocorticoids. Indeed, they showed that luteolin prevented bone mass loss, by downregulating apoptotic genes, increasing OPG/RANKL gene expression ratio, and activating the beta-catenin pathway in an animal model of osteoporosis induced by glucocorticoids. Therefore, these results suggest that this compound has an osteoprotective role in bone marrow.
On the other hand, apigenin is a phytoestrogen that has antiinflammatory effect on MSCs. Interestingly, apoptosis produced after the inflammatory response, triggered by lipopolysaccharide, was enhanced by the presence of this flavonoid. MSC differentiation is also influenced by apigenin. Different studies have suggested that it inhibits adipogenic differentiation of MSCs. Indeed, it has been found that apigenin downregulated PPARγ and inhibited fat droplet formation in 3T3-L1 cells that had been induced to differentiate into adipocytes. A negative effect of apigenin on adipogenesis via inhibition of the early differentiation processes, including mitotic clonal expansion, has also been reported. Yet, apigenin supplementation did not have effect in the advanced stages of adipogenic differentiation. Nevertheless, contrary to results obtained with mouse preadipocytes, this flavonoid did not inhibit adipogenesis in human MSCs derived from fatty tissue. Further studies are required to shed more light on putative roles of apigenin on human MSC differentiation into adipocytes.
The effect of apigenin on osteoblastogenesis has also been studied. This compound reduced apoptosis and ROS production, maintaining mitochondrial membrane potential, under oxidative stress conditions induced by H2O2 in MC3T3-E1 mouse preosteoblasts. Besides, its antioxidant activity maintained expression of osteoblastic genes in such cells and conditions. However, other authors have reported that this compound reduced viability and osteoblastogenesis in such cells, in a dose-dependent manner. Nevertheless, they reported that 10 mg apigenin/kg intraperitoneally administered to ovariectomized mice prevented loss of trabecular bone.
These in vivo results are in agreement with the ones obtained with human MSCs in vitro. For example, apigenin supplementation to osteogenic medium increased expression of osteoblastic markers and mineralization of extracellular matrix in human MSC cultures. Such effects activating osteoblastogenesis were mediated by increased phosphorylation of c-Jun N-terminal kinases (commonly known as JNKs) and p38 mitogen-activated protein kinases (commonly known as MAPKs). The role of p38 in MSC osteoblastogenesis has also been described. Interestingly, apigenin effects on MSCs may modulate bone metabolism, preventing bone mass loss in diseases like osteoporosis and physiological processes like aging.
The last important group of phenolics in extra virgin olive oil is the one of phenolic acids. These acids are simple phenols, usually found at very low concentrations in such food. Nevertheless, sometimes they are found at higher concentrations, like in some Tunisian olive oils, in which they can reach up to 38.39% of the total phenolic content. The most relevant phenols in this group include vanillic acid, syringic acid, 4-hydroxybenzoic acid, p-coumaric acid, and caffeic acid. Vanillic acid has hepatoprotective and cardioprotective effects[125,126]. Indeed, it has been observed that it reduced bone mass loss in ovariectomized mice. That may be related to its effects on cells derived from MSCs. For instance, this phenolic compound favored proliferation and differentiation of rat osteoblast-like UMR 106 cells, increasing expression of osteogenic markers and ratio of OPG/RANKL expression. These effects were mediated by its activity as phytoestrogen. It has also been suggested that vanillic acid may modulate adipogenesis because it reduced triglyceride content, without affecting cellular viability, in 3T3-L1 cells induced to differentiate into adipocytes.
On the other hand, syringic acid also has relevant healthy properties. Indeed, it is mainly a powerful antioxidant, being also antidiabetic, cardioprotective, antiinflammatory, etc. Yet, only its effect on MSC differentiation into osteoblasts have been studied. For example, it has been found that the presence of this compound in culture medium of mouse MSCs increased expression of osteogenic genes, alkaline phosphatase activity, and mineralization. This phenolic acid promoted osteoblastogenesis by inducing expression of the miR-21 microRNA, thus reducing expression of the so-called “mothers against decapentaplegic homolog 7” (SMAD7) gene, since it is the target of such microRNA. SMAD7 protein inhibits osteoblastogenesis. Therefore, its repression in MSCs promoted differentiation into osteoblasts[131,132]. In relation to 4-hydroxybenzoic acid, little is known about its putative effects on MSCs. Just one in vitro study has been carried out in relation to adipogenic differentiation of mouse 3T3-L1 and human MSCs from adipose tissue. Yet, in neither of these cell types did it produce significant changes during adipogenesis.
With respect to p-coumaric acid, it is usually found in extra virgin olive oil at concentrations lower than 1 mg/kg. Its intake is healthy, due to several biological activities, including antioxidant, antimicrobial, antiviral, antitumoral, antidiabetic, etc. Interestingly, extracts from the fragrant eupatorium plant (Eupatorium japonicum) favored osteoblastogenesis and inhibited adipogenesis in both multipotent C3H10T1/2 and primary bone marrow cells from mouse and rat, respectively. Besides, it prevented body weight increase and bone mineral density decrease in ovariectomized rats. Those effects were partially due to the biological activities of this phenolic compound. Similar results showed that p-coumaric acid inhibited adipogenic differentiation of 3T3-L1 preadipocytes[137,138]. Interestingly, this compound also inhibited myogenic differentiation of C2C12 cells from mouse, which may negatively affect development of skeletal muscle.
Finally, caffeic acid has antidiabetic and antioxidant activities. Yet, its putative biological activity on MSCs is unknown. Possibly, it can induce osteoblastic differentiation, since this compound increased alkaline phosphatase activity and changed the phenotype through a process involving antigen expression in human MG-63 osteosarcoma cells. Nevertheless, its mechanism of action and putative effects on undifferentiated cells remains unknown, requiring further research.
In summary, most studies about phenolic components of olive oil on MSCs have focused on how these compounds affect MSC differentiation into osteoblasts and adipocytes. In general, results have shown that such chemicals may modulate proliferation and differentiation of osteoblast precursor cells, suggesting that their consumption may influence bone health. The fact that phenolic extracts of extra virgin olive oil from different olive tree cultivars (Picual, Hojiblanca, Arbequina and Picudo) increased proliferation of MG-63 preosteoblastic cells supports that hypothesis. Indeed, olive oil consumption is associated with better bone health. That is partially due to the effects of its phenolic components on osteoblastic differentiation of MSCs. Therefore, it has been proposed that intake of this food, as well as other products derived from the olive tree, may help to prevent bone mass loss, due to diseases like osteoporosis[142,143] or physiological aging.