Polyphenol Antioxidants and Bone Health : A Review

Osteoporosis is a skeletal disease characterized by bone loss and structural deterioration of the bone tissue, leading to an increase in bone fragility and susceptibility to fractures, most frequently in the hip, wrist and spine (Sendur et al., 2009). Bone loss is associated with such factors as age, menopause in women, smoking, alcohol excess, calcium and vitamin D deficiency, low weight and muscle mass, anticonvulsant and corticosteroid use as well as certain co-morbid conditions such as rheumatoid arthritis (Javaid et al., 2008). Worldwide, it has been estimated that fractures caused by osteoporosis account for approximately one in three among women and approximately one in five among men over the age of 50. Although the mechanisms underlying osteoporosis are not fully understood, there is evidence suggesting that oxidative stress caused by reactive oxygen species (ROS) is associated with its pathogenesis (Sahnoun et al., 1997; Basu et al., 2001; Rao et al., 2007).


Introduction
Osteoporosis is a skeletal disease characterized by bone loss and structural deterioration of the bone tissue, leading to an increase in bone fragility and susceptibility to fractures, most frequently in the hip, wrist and spine (Sendur et al., 2009).Bone loss is associated with such factors as age, menopause in women, smoking, alcohol excess, calcium and vitamin D deficiency, low weight and muscle mass, anticonvulsant and corticosteroid use as well as certain co-morbid conditions such as rheumatoid arthritis (Javaid et al., 2008).Worldwide, it has been estimated that fractures caused by osteoporosis account for approximately one in three among women and approximately one in five among men over the age of 50.Although the mechanisms underlying osteoporosis are not fully understood, there is evidence suggesting that oxidative stress caused by reactive oxygen species (ROS) is associated with its pathogenesis (Sahnoun et al., 1997;Basu et al., 2001;Rao et al., 2007).
Oxidative stress is a condition that can be characterized by an imbalance of pro-oxidants and antioxidants with the scale being tipped towards an excess of pro-oxidants, creating abnormally high concentrations of ROS.ROS are a family of highly reactive, oxygencontaining molecules and free radicals, including hydroxyl (OH•-) and superoxide radicals (O2•-), hydrogen peroxide (H 2 O 2 ), singlet oxygen, and lipid peroxides (Juránek and Bezek, 2005).Several recent studies reported the impact of oxidative stress on osteoclast differentiation as well as on its function resulting to an increase in bone resorption (Garrett et al., 1990;Bax et al., 1992;Mody et al., 2001;Lean, 2003).Furthermore, recent in vitro studies have shown the important detrimental role of ROS on osteoblast activity (Park et al., 2005;Bai et al., 2004;Bai et al., 2005).In addition to in vitro and animal models, there is also increasing clinical evidence that oxidative stress might be involved in the pathogenesis of osteoporosis (Melhus et al., 1999;Sontakke & Tare., 2002;Basu et al., 2001;Maggio et al., 2003).
Antioxidants are known to mitigate the damaging effects of oxidative stress on cells.Epidemiological evidence has indicated a link between dietary intake of antioxidants and bone health.Fruits and vegetables are important sources of antioxidant phytochemicals that have been shown to play an important role in bone metabolism.Higher consumption of fruits and vegetables has been correlated with a reduction in the risk for the development of osteoporosis.(Arikan et al., 2011;Prentice et al., 2006;Macdonald et al., 2004;Macdonald et al., 2008;Palacios et al., 2006;Tucker et al., 1999;Lister et al., 2007;New, 2003;Trzeciakiewicz et al., 2009).Of particular interest among the antioxidant phytochemicals present in fruits and vegetables are the polyphenols.Polyphenols can be sub classified as non-flavonoids and flavonoids.Ellagic acid and stilbenes are among the major non-flavonoid polyphenols.Included in the flavonoid polyphenols are the anthocyanins, catechins, flavones, flavonols and isoflavones.The different categories of polyphenols, their chemical structures and sources are shown in Table 1.

Category
Numerous studies have shown the health-promoting properties of polyphenols, providing additional mechanisms through which they promote skeletal health by reducing resorption caused by high oxidative stress (Trzeciakiewicz et al., 2009;Tucker, 2009;Hunter et al., 2008).The antioxidant properties of polyphenols have been widely studied and reported in the literature (Liu et al., 2005;Miyamoto et al.,1998;Rassi et al., 2002;Viereck et al., 2002;Ward et al., 2001;Shen et al., 2011;Rao et al., 2007).They strongly support the role of polyphenols in the delayed onset or reduction in the progression of osteoporosis.The protective effects of polyphenols against diseases, including osteoporosis, have generated new expectations for improvements in health.This review will focus mainly on the role of polyphenols in osteoporosis and present results of studies undertaken in our laboratory.

Oxidative stress, antioxidants and osteoporosis
Oxidative stress occurs when the production of free radicals through a number of cellular events exceeds the ability of the cell's antioxidant defense to eliminate these oxidants (Baek et al., 2010).These free radicals have the ability to change the integrity of, and thus, damage several biomolecules, such as DNA, proteins and lipids (Baek et al., 2010).There is increasing evidence that oxidative stress is responsible for the pathophysiology of the aging process and may also be involved in the pathogenesis of atherosclerosis, neurodegenerative diseases, cancer, and diabetes.Recently, ROS were shown to be responsible for the development of osteoporosis (Sahnoun et al., 1997;Basu et al., 2001;Rao et al., 2007;Altindag et al., 2008;Becker, 2006;Feng & McDonald, 2011).Several in vitro and animal studies have shown that oxidative stress diminishes the level of bone formation by reducing the differentiation and survival of osteoblasts (Baek et al., 2010).Furthermore, it has been reported that ROS activate osteoclasts and thus, enhance bone resorption (Baek et al., 2010).The presence of ROS in osteoclasts was also demonstrated by Rao et al. in 2003 Recent evidences from a few clinical studies have also revealed that ROS and/or antioxidant systems might play a role in the pathogenesis of bone loss (Rao et al., 2007;Mackinnon et al., 2010;Abdollahi et al., 2005).
A number of studies have shown that antioxidants have a fundamental role in preventing postmenopausal osteoporosis.For instance, estrogens, whose antioxidant activity is essential in protecting women of reproductive age from cardiovascular disease, stimulate osteoblastic activity through specific receptors, thus favouring bone growth (Banfi et al., 2008).Antioxidant deficiency has been shown to have adverse effect on bone mass (Maggio et al. 2003).
Antioxidant enzymes are regarded as the markers of antioxidant defense mechanism against bone resorption.Several studies have investigated the relationship between antioxidant enzymes such as glutathione peroxidase (GP x ) and catalase (CAT) and osteoporosis (MacKinnon et al., 2011;Hahn et al., 2008;Maggio et al., 2003;Sontakke & Tare, 2002).
Recently, many dietary antioxidant nutrients have also been reported to decrease the oxidative stress that takes part in bone-resorptive processes (Rao et al., 2007;Weber, 2001;Peters & Martini, 2010;Macdonald et al., 2004).In addition to the antioxidant enzymes and nutrients, studies have also been directed towards the role of antioxidant phytochemicals such as the carotenoids in osteoporosis which will not be covered here, but has previously been reviewed (Rao & Rao, 2007;Sahni et al., 2009;Tucker, 2009).

Natural phytochemical antioxidants
Within the last decade, there has been an increased interest on polyphenols as a result of the in vitro evidence demonstrating that they may have numerous benefits to human health, mainly due to their antioxidative and free radical quenching properties (Hendrich, 2006;Lotito & Frei 2006;Heinonen, 2007;Stevenson & Hurst 2007;Aron & Kennedy 2008;Lopez-Lazaro, 2009;Saura-Calixto et al. 2007).It is therefore hypothesized that polyphenols may aid in the prevention of aging-associated diseases, particularly cardiovascular diseases, cancers, and osteoporosis.
Polyphenolic compounds are the products of the secondary metabolism of plant and are an essential part of human diet (Goldberg, 2003;Stevenson & Hurst 2007;D'Archivio et al., 2007;Saura-Calixto et al. 2007).To date, more than 8,000 polyphenols that have one common structural feature have been identified, a phenol, which is an aromatic ring possessing at least one hydroxyl substituent (Hendrich, 2006;Scalbert & Williamson, 2000;Harborne, 1993).The main classes of polyphenols include phenolic acids, flavonoids, stilbene, and lignans (Spencer et al., 2008;D'Archivio et al., 2007).Figure 1 illustrates the different groups of polyphenols, the chemical structures and food sources.Their total dietary intake can range up to 1 gram/day, which is considerably higher than that of all other classes of phytochemicals (Velioglu et al., 1998).There is much evidence demonstrating that polyphenols improve the status of different oxidative stress biomarkers.However, there is uncertainty regarding both the relevance of these biomarkers as predictors of disease risk and the appropriateness of the different methods used.

Modified resveratrol analogues
Myeloma cell lines U266 and OPM-2 Resveratrol analogues showed an up to 5,000-fold increased potency to inhibit osteoclast differentiation and promoted osteoblast maturation compared to resveratrol.

Polyphenols and osteoporosis
There has been an increase interest in the field of bone health and nutrients, and within the last decade, it has been well recognized that some polyphenols, whether ingested as supplements or with food, do in fact improve bone health status.Currently, most of the research on polyphenols and their effects has emerged from in vitro and in vivo studies with only a few clinical studies available.Compounds present in fruits and vegetables influence bone health as shown with in vitro osteoblast cell culture.On the other hand, epidemiologic studies tend to have mixed results with regards to the protective effects of polyphenol consumption against osteoporosis.Tables 2, 3, and 4 illustrate some of the recent in vitro, in vivo and clinical studies that have been reported in the literature, respectively.There have been several results suggesting that the combination of polyphenolic compounds found naturally in fruits and vegetables may reduce the risk of osteoporosis via increasing bone mineral density (Wu et al., 2002;Morton et al., 2001;Melhus et al., 1999;Leveille et al., 1997;Singh, 1992).In 1992, Singh was able to show that polyphenols afford protection against oxidative stress-induced bone damage during strenuous exercise.Similarly, Melhus was able to show its counteractive effect of polyphenols among smokers (Melhus et al., 1999).

Research results on the role of polyphenols in osteoporosis from the author's laboratory
Previous in vitro results from our laboratory have shown that a supplement rich in a variety of polyphenols commercially known as greens+ TM , is more effective in stimulating osteoblasts to form more bone nhodules in a dose-dependant manner than epicatechin, the main polyphenol found in green tea (Fig. 2).Our laboratory also studied the effects of a second supplement, bone builder TM , which is rich in minerals, vitamins and nutrients.
Similarly to the greens+ TM , the water-soluble bone-builder extract had a significant dosedependent stimulatory effect on bone nodules formation (Fig. 3). Figure 4 shows that when the two supplements, greens+ TM and bone builder TM , were tested as combination, the effects were six times more effective than either one alone.This led us to believe that synergistic effects of greens+ TM and bone builder TM may have a beneficial effect on osteoporosis.We then conducted a clinical evaluation of this nutritional supplement greens+ bone builder TM Results have shown that there was an increase in total antioxidant capacity after 8 weeks of treatment compared to placebo (Fig 4).as well as a decrease in both lipid and protein oxidation over a 4 and 8-weeks of intervention with greens+ bone builder TM compared to placebo (Fig. 6 & 7).This suggests that the nutritional supplement may have a beneficial effect on bone health by mitigating the effects of oxidative stress.

Conclusions
Although epidemiologic studies are practical for the evaluation of human health effects on the physiologic concentrations of polyphenols, reliable data on polyphenol contents of foods are limited.This review has shown that polyphenols or polyphenol-rich diets can provide significant protection or treatment for the development and progression of osteoporosis.Keeping in mind that many nutrients are co-dependent, and they may interact among themselves and others.The complexity of these interactions may possibly be the reason why many studies show controversial or inconsistent results regarding the effects of a single nutrient or groups of nutrients in bone health.Based on current knowledge, polyphenols offer a platform for the prevention of many human chronic diseases involved with oxidative stress, including osteoporosis.
To value the actual significance of food phenolics, it is necessary to investigate not only their bioavailability, but also their mechanisms of action and their possible synergism with other constituents either in the diet or within the human body, as well as the polyphenolic content and composition of foods.We have attained this goal by studying the nutritional supplement greens+ TM , which is rich in polyphenols and their interactions with minerals, vitamins and nutrients that were present in the nutritional supplement bone builder TM .

Fig. 1 .
Fig. 1.The role of oxidative stress in osteoporosis and how/where antioxidants play a role in mitigating ROS

Fig. 7 .
Fig. 7. Change in protein oxidation over 4 and 8-weeks of nutritional intervention with g+bb TM compared to placebo (p<0.05).

Table 1 .
The different categories of polyphenols, their chemical structures and sources

Table 2 .
Polyphenols-In vitro studies www.intechopen.comPhytochemicals -A Global Perspective of Their Role in Nutrition and Health 472

Table 3 .
Polyphenols-In vivo Studies