Keywords

Key Facts

Key Facts on In Vitro Effects of Soy Phytoestrogens on Bone Cell

  • Soy phytoestrogens suppress the formation of osteoclast.

  • Soy phytoestrogens promote proliferation and differentiation of osteoblast.

  • Osteoblastic differentiations are induced by expressions of BMP, Col I, and OCN genes as well as p38 MAPK-Cbfa1 and estrogen receptor-protein kinase C alpha (ER-PKCα)-related signaling pathways.

  • Osteoclastic differentiation is induced by suppression of NF-kB (RANKL).

Key Facts on In Vivo Effects of Soy Phytoestrogens in Ovariectomized (OVX) Rats

  • Soy phytoestrogens prevent bone loss in OVX rats which represent the condition of postmenopausal estrogen deficiency.

  • Soy phytoestrogens increase femoral mass as well as both tibia and femur BMD in OVX animals.

  • The activity of soy phytoestrogen is enhanced in the presence of other supplements such as vitamin, soy extract, and soy yogurt.

Key Facts on In Vivo Effects of Soy Phytoestrogens in Intact and Orchidectomized (ORX) Rats

  • Soy protein without isoflavone enhances bone quality in ORX rats.

  • Soy isoflavones show effects on Tb.Sp, trabecular number, and BV/TV in ORX rats.

  • Soy phytoestrogens exhibit positive effects on bone health in in utero and intact rats.

  • Soy phytoestrogens show no effects on bone heath of intact rats as well as on the lactation period in female rats.

Key Facts on Effects of Soy Phytoestrogens in Postmenopausal Women

  • Soy phytoestrogens exhibit conflicting results in human clinical studies.

  • No effects on bone loss and bone turnover.

  • Increases bone formation and reduces bone resorption in few studies.

  • Shows positive effects on BMD in few studies.

  • No effects on bone marker level on bone biomarkers such as bone-specific alkaline phosphatase (BAP) and N-telopeptide of type 1 collagen (NTx)/creatinine.

Key Facts on Effects of Ipriflavone, a Synthetic Isoflavone in In Vitro, In Vivo, and Human Studies

  • IP is derived from soy isoflavone daidzein.

  • IP increases bone formation and inhibits bone resorption in animal and human bone cells.

  • IP maintains bone mineral content, restores bone mass, and increases bone or bone marrow percentage in animal models.

  • IP prevents bone loss and promotes bone formation in postmenopausal women.

Introduction

Osteoporosis is defined as a condition of low mineral density resulting in fragile bones with increased risk of fracture (Bernabei et al. 2014). The World Health Organization (WHO 1994) defines osteoporosis as a bone mineral density less than 2.5 standard deviations (SD) below the standard reference for maximal bone mineral density of a young adult female. Women are more prone to develop osteoporosis as compared to men due to the decrease in estrogen level after menopause leading to the decline in bone formation and increase in bone resorption activity (Roush 2011). However, male osteoporosis is becoming an increasingly important public health problem (Gielen et al. 2011). One in three osteoporotic fractures occurs in men from age 50 onward and fracture-related morbidity and mortality are even higher than in women (Gielen et al. 2011). Hormone replacement therapy (HRT) is widely used in the prevention and treatment of osteoporosis. However, HRT has considerable side effects, such as increased risks of breast cancer, uterine cancer, and thromboembolism (Ferguson 2004). According to Women’s Health Initiative studies, participants on HRT had slightly higher rates of breast cancer, ovarian cancer, heart attack, stroke, thromboembolism, and Alzheimer’s disease compared to nonusers (Rossouw et al. 2002; Chlebowski et al. 2003; Shumaker et al. 2003). The problems associated with HRT lead to the development of alternative therapeutics in the management of osteoporosis incorporating phytoestrogens (Brink et al. 2008).

Phytoestrogens are polyphenolic compounds that structurally and functionally mimic the endogenous estrogen, 17β-estradiol (E2), which are broadly classified into three main groups, isoflavones, lignans, and coumestans (Dixon 2004). Soybean (Glycine max, Fabaceae) food contains macronutrients such as lipids, carbohydrates, and proteins and micronutrients such as isoflavones, phytate, saponins, phytosterol, vitamins, and minerals (Cederroth and Nef 2009). Soybeans are rich in isoflavones and have been widely used as a dietary source of phytoestrogens in animal and human studies (Cederroth and Nef 2009). The metabolism of isoflavones is complex. Two major isoflavones present in soybeans as β-D-glycosides, namely, genistin and daidzin (Fig. 1), are biologically inactive (Setchell 1998). Once ingested, these glycosides are hydrolyzed in the intestinal tract by bacterial β-glucosidases forming the corresponding bioactive aglycones, genistein, and daidzein, which are absorbed into the bloodstream. Daidzein can be further metabolized in the digestive tract to dihydrodaidzein, equol and o-desmethylangolensin (O-DMA), and genistein to p-ethyl phenol (Setchell 1998). Isoflavones in soybeans are tightly bound to proteins, which explains the variability of phytoestrogen contents in different soy products and therefore their availability for absorption in the digestive tract. Bhathena and Velasquez (2002) reported the soy protein contents in different soy products as follows: 0.1–5 mg isoflavones/g of soy protein in mature and roasted soybeans, 0.3 mg/g soy protein in green soybeans and tempeh, and 0.1–2 mg/g soy protein in tofu and selected soy milk preparations.

Fig. 1
figure 1

Soy phytoestrogens and metabolism. The above diagram shows active forms of soy phytoestrogens and their metabolic derivatives

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Genistein , daidzein, equol, and O-DMA are the major isoflavones detected in blood and urine of humans and animals (Setchell 1998). In rodents, equol is the major circulating metabolite representing up to 70–90% of all circulating isoflavones. While all rodents are equol producers, only 30% of humans are able to metabolize daidzein into equol (Atkinson et al. 2005). Pharmacokinetic studies confirm that healthy adults absorb isoflavones rapidly and efficiently (Setchell et al. 2001). The average time for the aglycones in phytoestrogen-rich food to reach plasma concentrations after ingestion is 4–7 h. Hydrolysis of glycosidic moiety of β-glycosides in phytoestrogen-rich food is a rate-limiting step for absorption since it can delay absorption of aglycones to 8–11 h (Setchell et al. 2001).

This review aims to present a brief summary of the role of soy phytoestrogen and its synthetic derivative, ipriflavone (IP), on biomarkers of osteoporosis primarily based on studies using murine and human bone cells, experimental animal models, and human studies (Fig. 2).

Fig. 2
figure 2

Effects of soy phytoestrogens and ipriflavone on biomarkers of osteoporosis in experimental models and in humans . The above figure is a summary of the effects of soy phytoestrogens and ipriflavone on bone metabolism and biomarkers of osteoporosis using cultured murine or human bone cells, animals (intact, ovariectomized, orchidectomized), and humans (postmenopausal women)

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In Vitro Effects of Soy Phytoestrogens on Bone Cell Metabolism and Biomarkers of Osteoporosis

Bone remodeling is defined as the removal of mineralized bone by osteoclast s followed by the formation of bone matrix by osteoblasts , which subsequently becomes mineralized (Hadjidakis and Androulakis 2006). The remodeling cycle consists of three consecutive phases: resorption, reversal, and formation. During resorption, partially differentiated mononuclear preosteoclasts migrate to the bone surface where they form multinucleated osteoclasts. After completion of osteoclastic resorption, in the reversal phase, mononuclear cells provide signals for osteoblast differentiation and migration and prepare the bone surface for new osteoblasts to begin bone formation. In the formation phase, osteoblasts lay down new bone completely replacing the resorbed bone (Hadjidakis and Androulakis 2006). At the end of the remodeling cycle, the bone surface is covered with flat lining cells and rests for a period of time before the next remodeling cycle (Hadjidakis and Androulakis 2006). Table 1 summarizes the effects of soy phytoestrogens on in vitro murine and human osteoblasts, osteoblast-like cells, osteoclasts, and bone marrow stromal osteoprogenitor cells (BMSCs). In general, the regulation of bone remodeling is both systemic and local. The major systemic regulators include parathyroid hormone (PTH), calcitriol, and other hormones such as growth hormone, glucocorticoids, thyroid hormones, and sex hormones. A large number of cytokines and growth factors that affect the bone cell function are attributed to the local regulation of bone remodeling (Hadjidakis and Androulakis 2006). Examples of growth actors are insulin-like growth factor (IGFs), prostaglandins, tumor growth factor-beta (TGF-β), and bone morphogenetic protein (BMP). Furthermore, through the receptor activator of NF-kappa B ligand/osteoprotegerin (RANKL/OPG) system, the processes of bone resorption and formation are tightly coupled, thus maintaining the skeletal integrity through the bone formation followed by each cycle of bone resorption (Hadjidakis and Androulakis 2006). Besides effects on systemic and local regulators, Table 1 also summarizes the effects of soy phytoestrogens through estrogen receptor (ER) and non-estrogen receptor (non-ER) pathways on bone remodeling. Studies by Chen and Wong 2006, Liao et al. 2014, Strong et al. 2014, and Wang et al. 2014 reported that osteoblastic differentiation in corresponding human and murine bone cells is mediated through the ER pathways. Human osteoblasts express both estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), although the expression of ER subtypes varies during differentiation (Onoe et al. 1997; Bodine et al. 1998). The greatly increased expression of ERβ during bone mineralization (Arts et al. 1997) is particularly pertinent to the potential hormonal effects of phytoestrogens since compounds such as genistein show a much higher affinity for ERβ than for ERα (Kuiper et al. 1998; Morito et al. 2001). Osteoblastic differentiation is also mediated through mitogen-activated protein kinase-core binding factor 1 (MAPK-Cbfa1) and other non-ER pathways (Liao et al. 2007; Strong et al. 2014). Osteoblastic differentiation and inhibition of osteoclastic formation are modulated by prevention of nuclear factor kappa B (NF-kB) translocation (Lee et al. 2014).

Table 1 In vitro effects of soy phytoestrogens on bone cell metabolism and biomarkers of osteoporosis
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In Vivo Effects of Soy Phytoestrogens on Bone Metabolism and Biomarkers in Ovariectomized (OVX) Rats

In vitro studies provide insight into the effects of soy phytoestrogens on individual cells, whereas in vivo studies provide the advantage of using intact systems that take into account coupling effects among osteoblast s, osteoclasts , their progenitor cells, and the effects of metabolic activities that influence the efficacy of a compound (Setchell and Lydeking-Olsen 2003). Table 2 summarizes the effects of soy phytoestrogens in ovariectomized (OVX) rats. Due to acute deficiency of ovarian estrogen that leads to loss of bone mass, the OVX rat represents a good postmenopausal osteoporosis model (Setchell and Lydeking-Olsen 2003). In rats, ovariectomy leads to a selective reduction in the number of vitamin D receptors (VDR) in jejunum (Chan et al. 1984). This reduction in VDR results in lower responsiveness of intestinal cells to vitamin D signaling and lower calcium absorption by the intestine. This leads to reduction in bioavailability of dietary calcium, an essential building block for new bone formation. Arjmandji et al. (1996) reported that soy protein isolate was as effective as estradiol in retarding bone loss following OVX in rat model. Devareddy et al. (2006) observed that treatment of OVX rats with soy isoflavones did not increase the tibial bone mineral density (BMD) up to the level of sham despite a small percentage (4.5%) of increase in BMD as compared to the OVX controls. The soy isoflavone treatments also did not show any beneficial effects on lumbar microarchitectural properties in OVX rats (Devareddy et al. 2006). Om and Shim (2007) and Rachon et al. (2007) reported the positive effects of purified soy phytoestrogens daidzein and equol on OVX rats. Daidzein increased the femoral mass in cadmium-induced OVX rats (Om and Shim 2007) and equol attenuated trabecular bone loss and increased the density of lumbar spine in OVX rats (Rachon et al. 2007). Other studies (Shiguemoto et al. 2007; Jeon et al. 2009; Zhang et al. 2009; Chang et al. 2013) reported that diet supplemented with isoflavone and vitamin D resulted in high BMD and alkaline phosphatase (ALP) activity and maintained the proper bone microarchitecture indicating the bone-sparing effects of soy phytoestrogens on OVX rats.

Table 2 In vivo effects of soy phytoestrogens on bone metabolism and biomarkers in ovariectomized (OVX) rats
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In Vivo Effects of Soy Phytoestrogens on Bone Metabolism in Intact and Orchidectomized (ORX) Rats

Osteoporosis poses a great challenge to the aging population in the USA, and though largely manifested in women, men also exhibit risk factors of this degenerative condition (Khosla 2010). One-third of hip fractures and one-half of symptomatic vertebral fractures are reported in men (Johnell and Kanis 2006). One of the causes for male osteoporosis is hypogonadism with aging (Becker 2008; Szulc et al. 2001; Khalil et al. 2005; Soung et al. 2006). Table 3 summarizes the effects of soy phytoestrogens on orchidectomized (ORX) and intact rat models. Few studies (Khalil et al. 2005; Soung et al. 2006; Juma et al. 2012) have focused on the effects of soy phytoestrogens on ORX rats, and even fewer studies (James et al. 2002) have concentrated on the effects on soy phytoestrogens on young and peripubertal rats.

Table 3 In vivo effects of soy phytoestrogens on bone metabolism in intact and orchidectomized (ORX) rats
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James et al. (2002) aimed to compare calcium metabolism and bone mineralization in young female rats after feeding them casein versus isoflavone-rich diets. Results indicated that compared to soy protein, casein either alone or with the addition of isoflavones showed positive effects on growth and bone mineralization in the peripubertal period when the growth rate was at its maximum. Results also indicated that calcium metabolism was higher in casein with isoflavone-treated rats compared to soy protein. Studies on intact rats yielded mixed results detailed in Table 2. For example, a pilot study by Peterson et al. (2009) demonstrated that bone formation in female Wistar rats during lactation was not effected by soy isoflavone consumption. Another study by Nakai et al. (2005) showed that soy protein and isoflavones have no effects on femur and lumbar BMD of intact Sprague–Dawley female rats. The study by Ward and Piekarz (2007) indicated that genistein, daidzein, or their combinations have no effect in utero and femur peak load. The positive effects of cladrin were reported on female Sprague–Dawley rats (Gautam et al. 2011). Cladrin increased the mineral apposition (MAR) and bone formation rates compared to controls, whereas formononetin showed no effect on bone formation in vivo (Gautam et al. 2011).

Effects of Soy Phytoestrogens on Biomarkers of Osteoporosis in Postmenopausal Women

Postmenopausal estrogen deficiency results in increased bone resorption, which is the major contributing factor of osteoporosis (Leboime et al. 2010). Bisphosphonates, HRT, and other antiresorptive treatments are available for the treatment and even prevention of postmenopausal osteoporosis. However, HRT has been associated with health problems such as coronary heart disease, pulmonary embolism, and stroke (Rossouw et al. 2002), whereas the use of bisphosphonate can lead to osteonecrosis of the jaw and atypical fractures (Arrain and Masud 2008). These adverse side effects have led to the identification and use of complementary and alternative treatments, which are considered safer and effective (Barnes et al. 2008). Phytoestrogens, especially isoflavones, have been used as dietary alternatives to HRT and Food and Drug Administration (FDA)-approved drugs (alendronate, risedronate, ibandronate, zoledronic acid, raloxifene, denosumab) (Pawlowski et al. 2015). Soybean isoflavones, components of dietary supplements, are genistein, daidzein, and glycitein. Setchell et al. (2002) reported that subjects who have gut microflora that can metabolize daidzein to equol showed greater activity to isoflavones than those who do not have the proper microflora. Table 4 summarizes the effects of soy isoflavones and soy food on postmenopausal bone loss. Results of the studies mentioned in Table 4 show variability regarding the efficacy of isoflavones in preventing postmenopausal bone loss. Some studies (Morabito et al. 2002; Chen et al. 2003; Marini et al. 2007; Pawlowski et al. 2015) show positive bone-sparing effects of phytoestrogens, whereas others (Brink et al. 2008; Alekel et al. 2010; Tai et al. 2012) show no effects on reducing bone loss. These result differences could be attributed to the differences in population under study, sample size, and study duration. In addition to human clinical studies, epidemiology also revealed the protective effects of soy phytoestrogens in women against osteoporosis (Somekawa et al. 2001; Zhang et al. 2005). Thus, while some studies are promising, further research is needed on the effects of whole soy foods, soy proteins, and purified isoflavones in larger trials to support the beneficial effect of soy phytoestrogens in osteoporosis.

Table 4 Effects of soy phytoestrogens on biomarkers of osteoporosis in postmenopausal women
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Effects of Ipriflavone, a Synthetic Isoflavone on Bone Biomarkers in In Vitro, In Vivo, and Human Studies

Evaluation of phytoestrogens, mainly soy isoflavones, as candidates for bone loss treatment are also supported by results on the bone-sparing effects of ipriflavone (IP) (Brandi 1993). Ipriflavone, 7-isopropoxyisoflavone, is a synthetic isoflavone derived from daidzein in the 1930s (Sziklai et al. 1992; Head 1999) showing positive effects in the treatment and prevention of osteoporosis by suppressing bone resorption, increasing bone calcium retention and enhancing the beneficial action of estrogen on bone metabolism (Reginster 1993). Ipriflavone has been used as an alternative to HRT in the prevention of acute bone loss in postmenopausal women (Reginster 1993). Arjmandi et al. (2000) reported that IP prevents bone loss in postmenopausal women and OVX rats, and Ge et al. (2010) observed the significant effect of IP in increasing BMD, osteocalcin, and hydroxyproline contents in a dose-dependent manner in OVX rats. Ipriflavone is extensively metabolized in the liver and excreted in urine. In dogs and rats, seven metabolites were identified in the plasma. However, in humans, only MI, MII (daidzein), MIII, and MV seem to predominate. Out of these metabolites, MIII is the most potent than MII and MI and MV were least potent (Head 1999). Table 5 summarizes selected in vitro, in vivo, and human studies on the effects of ipriflavone on osteoporosis. Ipriflavone stimulated osteoblast and inhibited osteoclast formation in murine and human bone cells (Giossi et al. 1996; Yao et al. 2007; Civitelli 1997). In vivo effects of IP were observed in caged hens (Yao et al. 2007; Lv et al. 2014) where IP increased egg production while maintaining the bone mineral content and alleviated caged layer osteoporosis (CLO). Ipriflavone also increased bone formation and restored bone mass in male Japanese white rabbits and Sprague–Dawley rats, respectively (Minegishi et al. 2002; Deyhim et al. 2005). According to Zhang et al. (2010), ipriflavone exhibited positive effects in postmenopausal women by inhibiting bone resorption, whereas Alexandersen et al. (2001) and Katase et al. (2001) reported no effects of IP in postmenopausal bone loss and biochemical markers of bone metabolism.

Table 5 Effects of ipriflavone, a synthetic isoflavone on bone biomarkers in in vitro, in vivo, and human studies
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Potential Applications to Prognosis, Other Diseases, or Conditions

As the human population ages, osteoporotic fractures are increasingly recognized as a common and serious health problem that significantly compromise quality of life in elderly people. Osteoporosis and its consequence of fragility fractures are characterized by highly complex phenotypes, which include BMD, bone strength, bone turnover markers, and nonskeletal traits, as reviewed earlier in this chapter. Thus, the early identification of bone biomarkers that are associated with osteoporosis phenotypes or response to therapy can eventually help individualize the prognosis, treatment, and prevention of fractures and their adverse outcomes. Bone density assessment has been identified as a clinically useful and cost-effective tool in the prognosis and treatment of osteoporosis in older adults (Schousboe et al. 2005, 2007). Although BMD is well established as a predictor of future fracture risk and several prospective studies have demonstrated a 1.5- to 2.5-fold increased risk of fracture for every 1 SD decrease in BMD, this biomarker alone displays poor sensitivity in predicting future fractures. Thus, fracture risk assessment scores are better used in the prognosis of osteoporosis. One such example is the WHO Fracture Risk Assessment Tool (FRAX®), which combines age and sex with clinical risk factors to provide an estimate of the 5- or 10-year probability of fracture for an individual (Kanis et al. 2008). A clear advantage of fracture prediction tools is that they provide an estimate of absolute risk, in that if a 55-year-old woman has osteoporosis according to dual-energy X-ray absorptiometry (DXA), for example, she can still have a low 10-year risk of fracture that might not indicate the need for pharmacological treatment. The estrogenic effects of isoflavones have led researchers to view soy foods and isoflavone supplements as alternatives to conventional hormone therapy. However, as described earlier in this chapter, the evidence that isoflavones reduce bone loss in postmenopausal women is quite conflicting and can be largely explained by the heterogeneity in the study sample and dosing and overall limited clinical studies in this area. Based on the anabolic effects of soy phytoestrogens on bone formation in preclinical animal models of osteoporosis, the inclusion of whole soy foods and beverages may be considered a positive health choice in the older population. Further studies must identify the effects of soy phytoestrogens on novel bone biomarkers, such as those related to genomics, epigenomics, and metabolomics, as well as composite fracture risk scores in the prognosis and management of osteoporosis.

Summary Points

  • Soy phytoestrogens promote osteoblastic differentiation through the expressions of biomarkers such as: BMP (participates in matrix differentiation and bone formation), collagen type 1(Col I) (stimulates osteoblast adhesion and differentiation), and osteocalcin (OCN) genes (control osteoblast function).

  • Soy phytoestrogens suppress osteoclast formation by inhibiting translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) (transcription factor and essential receptor activator for RANKL-induced osteoclast formation) to the nucleus.

  • In postmenopausal (OVX) rat models, soy phytoestrogens increase femoral mass and BMD in the tibia and femur.

  • In male osteoporosis represented by ORX rats, soy isoflavones show effects on trabecular separation (Tb.Sp), trabecular number, and trabecular bone volume (BV/TV).

  • Soy isoflavones increase bone formation, reduce bone resorption, and exhibit positive effects in studies in postmenopausal women, whereas in few studies soy isoflavones show no effects on bone biomarkers such as bone-specific alkaline phosphatase (BAP) and N-telopeptide of type 1 collagen (NTx)/creatinine.

  • Ipriflavone, the synthetic isoflavone, shows positive effects in cultured bone cells, animals, and postmenopausal women.

Definitions of Words and Terms

Alkaline phosphatase (ALP):

An enzyme that hydrolyzes phosphate esters and liberates inorganic phosphate with an optimal pH of about 10.0 serum ALP activity increases in bone diseases such as bone cancer, hyperparathyroidism, and osteitis deformans

Bone marrow-derived mesenchymal stem cell (BMSC):

Postnatal stem/progenitor cells capable of self-renewing and differentiating into osteoblasts, chondrocytes, adipocytes, and neural cells

Bone mineral density (BMD):

Measurement of calcium in the bone which indicates the strength of bone

Bone morphogenetic protein (BMP):

30–38-kD homodimeric family of protein involved in the formation of bone and cartilage and provides morphogenetic signals guiding normal tissue architecture

Lymphocytopenia:

A condition with abnormally low levels of blood lymphocytes

Orchidectomized rat:

Male rats with one or both testicles removed

Ovariectomized rat:

Female rats with one or both ovaries removed

Peripubertal:

Early stages of puberty

Postmenopausal:

Time period after which a woman undergoes a lack of menstruation for twelve consecutive months

Trabecular bone volume (BV/TV):

The fraction of a given volume of interest (VOI) occupied by mineralized bone. It is reported as a % value and is also used to evaluate a bone volume density following a given treatment

Trabecular number (Th.N):

Quantification of relative number of individual trabeculae within 3-D region of interest (ROI). It is also one of the bone microstructural indices

Trabecular separation (Tb.Sp):

Quantification of relative spacing between individual trabeculae within 3-D ROI. It is one of the bone microstructural indices