4.

Martnez OG, Rivas A, Torrecillas JR, Bertos EDL, and Ruiz C. The effect of
olive oil on osteoporosis prevention. Int J Food Sci Nutr. 2014, Early Online: 17
The World Health Organization (WHO) defines osteoporosis (OP) as a
progressive systemic skeletal disease characterized by bone mass loss and
microarchitectural deterioration of bone tissue that increases bone fragility and the
risk of fracture, the main clinical squealed of the disease. The high-morbidity and
mortality and elevated economic costs associated with these fractures led the WHO to
describe OP as the second most important healthcare problem worldwide after
cardiovascular disease. The prevalence of OP is currently 20% and may increase with
the progressive aging of populations.
OP development is influenced by multiple factors. Bone modeling and remodeling processes are governed not only by heritable traits but also by nutritional,
mechanical and hormonal factors. Nutrition has various relevant effects on bone mass
peak, age related bone loss and muscle strength, among others. The main nutrients of
interest in this respect are calcium and vitamin D, due to their role in optimizing the
bone mass peak. However, the European Union has acknowledged the relevance of
other nutrients and has called for further research on their effects on bone. Although
nutrition is only one of many factors affecting the amount and fragility of bone mass,
it is of particular importance to bone health because it is modifiable.
The so-called Mediterranean diet (MeDi) is characterized by a high intake in
fruits, vegetables and olive oil. The incidence of osteoporosis and associated fractures
is found to be lower in countries where the MeDi is predominant. These observations
might be mediated by the active constituents of virgin olive oil (VOO) and especially
phenolic compounds. Therefore, the objective of this study was to review
publishedreports on the effect of VOO and its phenols on bone health.

Phenolic compounds of virgin olive oil and bone mass Besides triglycerides,
VOO contains a wide variety of so-called minor compounds that are of major
importance from a chemical and organoleptic standpoint. These include phenolic
compounds, which constitute a highly complex fraction formed by numerous
substances, some of which are yet to be identified. The known substances include
simple phenols, e.g. hydroxytyrosol, tyrosol, caffeic acid, vanillic acid, p-coumaric
acid, ferulic acid, and vanillin; flavonoids, e.g. luteolin and apigenin; and other more
complex compounds such as those derived from oleuropein, ligstroside, ligustaloside,
verbascoside and lignans.
Oleuropein is the main phenolic compound in olive-tree leaves, olives and
olive oil, with the amount found in olives and olive oil ranging between 1 ppb and 11
ppm. Oleuropein was found to enhance bone health by increasing the formation of
osteoblasts from bone marrow stem cells and decreasing the generation of adipocytes
or fat cells, suggesting that oleuropein intake might have preventive effects against
the bone loss associated with osteoporosis and aging.
Puel et al. (2004) evaluated the effect of oleuropein in a model of
ovariectomized rats with and without inflammation. Ovariectomised rats received diet
with 0.15 g oleuropein/kg/day. This phenolic compound was able to elicit protective
effects on bone loss in this model, probably by modulating variables of inflammation
(such as a-1-acid glycoprotein). However, it had no effect on bone mineral density
when inflammation was not performed. In addition, Puel et al. (2006) found in the
same model that the administration of oleuropein at doses ranging between 2.5 and 15
mg/Kg/day for 100 days reduced the bone mass loss, attributing this effect to its
modulation of inflammatory biomarkers.
In particular, hydroxytyrosol has been identified as one of the most potent
antioxidants found in olive oil. Hydroxytyrosol at 10 to 100 mM had no effect on the
production of type I collagen and the activity of alkaline phosphatase in MC3T3-E1

cells, but stimulated the deposition of calcium. In contrast, hydroxytyrosol at 50 to

100 mM inhibited the formation of multinucleated osteoclasts in a dose-dependent
manner. Furthermore, hydroxytyrosol suppressed the bone loss of trabecular bone in
femurs of ovariectomized mice (6-week-old BALB/c female mice).
Puel et al. (2008) showed that tyrosol and hydroytyrosol administration in the
diet at doses of 0.017% prevented the inflammation-induced bone mass loss in
ovariectomized rats in a model of senile osteoporosis and increased their osteoblast
activity, as evidenced by their elevated plasma osteocalcin levels. This bone-sparing
effect did not seem to be ascribable to an improvement of the inflammatory state,
given that the increase of inflammatory parameters tested in this investigation was not
prevented, but rather to their antioxidant properties.
Finally, flavonoids, a group of phenolic compounds that are abundant in
plants, are of great interest due to the multiple biological effects obtained via their
anti-inflammatory, antioxidant and estrogenic activities. Luteolin, a flavonoid present
in VOO, markedly decreased the differentiation of both bone marrow mononuclear
cells and Raw 264.7 cells into osteoclasts. It also inhibited the bone resorptive
activity of differentiated osteoclasts. The effects of luteolin on ovariectomy-induced
bone loss using microcomputed tomography, biomechanical tests and serum markers
assay for bone remodeling was investigated. Oral administration of luteolin (5 and 20
mg/kg per day) to ovariectomized mice caused significant increase in bone mineral
density and bone mineral content of trabecular and cortical bones in the femur as
compared to those of ovariectomized controls, and prevented decreases of bone
strength indexes induced by ovariectomy surgery. Serum biochemical markers assays
revealed that luteolin prevents ovariectomy-induced increases in bone turnover. These
data strongly suggest that luteolin has the potential for prevention of bone loss in
post-menopausal osteoporosis by reducing both osteoclast differentiation and
function.

Mechanism of action of phenolic compounds present in olive oil on bone

Oxidative stress is a pivotal pathogenic factor for age-related bone loss in
mice and rat, leading to an increase in osteoblast and osteocyte apoptosis, among
other changes, and a decreased in osteoblast numbers and in the rate of bone
formation. Several lines of evidence suggest a tight association between oxidative
stress and the pathogenesis of osteoporosis in humans. VOO phenolic compounds
have been shown to possess antioxidant properties in vivo and in vitro. The intake of
phenols may influence bone mineral density by acting as free radical scavengers,
preventing oxidation-induced damage to bone cells. In addition, the growing
understanding of the bone remodeling process supports the theory that inflammation
significantly contributes to the etiopathogenesis of osteoporosis. It has been showed
that baseline markers of inflammation were higher among subjects who subsequently
experienced and incident fracture. Besides their widely reported antioxidant and free
radical neutralizing activities, there is evidence that some of VOO phenols exert antiinflammatory activity and can influence various processes, including cell
differentiation, cell integrity preservation, maintenance of DNA repair mechanisms
and induction of cancer cell apoptosis.
It has been suggested that VOO phenolic compounds may provide desirable
bone health benefits through an action on bone cell metabolism. Bone metabolic
diseases, like OP, develop when there is an imbalance between the formation and
resorption of bone, which depend on the interaction between osteoblasts and
osteoclasts. Numerous cytokines, hormones, and growth factors control bone
formation by regulating osteoblast cell proliferation and differentiation in which
differentiated osteoblast phenotype is expressed, including genes that encode for ALP
and osteocalcin, producing a high level of ALP synthesis.

It has been demonstrated that different phenolic compounds in vegetable

species (e.g. resveratrol in wine or quercetin in grape or onion) can modulate
osteoblastic functions, such as their proliferative capacity or cell maturation, by
increasing ALP activity and calcium ion deposition in the extracellular matrix.
Santiago-Mora et al. (2011) showed that oleuropein, increases the expression of
Runx2 and osterix, indicating that this phenol enhances osteoblast formation in both
initial and later phases of differentiation. The role of oleuropein in osteoblast
differentiation was supported by the higher expression levels of other osteoblast
markers, such as ALP gene expression/activity, expression of different biomarkers
(CD54, CD80, CD86 or HLA-DR) or collagen type I gene expression after exposure
to this phenol. In addition, Banchereau et al. (2000) demonstrated that the presence of
ferulic or caffeic acid produced a major increase in the expression of CD86 and HLADR, which may be associated with a state of cell activation, given that their
expression has been related to the degree of differentiation and/or cellular activation.

Bioavailability of olive oil phenolic compounds in humans

The degree to which VOO phenolic compounds are bioavailable is
fundamental in understanding and evaluating the bone health benefits associated with
such compounds. Research on humans and animals have shown that the dominant
VOO phenols, hydroxytyrosol and tyrosol, are bioavailable and, as such, a fraction of
the oral dose can be recovered in urine, which are mainly glucoronoided conjugates.
Corona et al. (2006) conducted a detailed investigation into the absorption,
metabolism and microflora- dependent transformation of the VOO phenolic
compounds hydroxytyrosol, tyrosol and their conjugated forms, such as oleuropein.
The conjugated forms underwent rapid hydrolysis under gastric conditions, resulting
in significant increases in the amount of free hydroxytyrosol and tyrosol entering the

small intestine. In addition, Mateos et al. (2011) showed that hydroxytyrosol was
transferred across human Caco-2/TC7 cell monolayers. A further study found that the
absorption of administered hydroxytyrosol, and tyrosol was as high as 5566% of the
ingested phenols in humans, indicating an effective intestinal absorption of these
compounds.

Mechanism of action of phenolic compound present in olive oil on osteoblasts.