Menopause is a major health concern for women, as it increases susceptibility to various chronic diseases, including cardiovascular diseases (CVDs), osteoporosis, arthritis, Alzheimer's disease, obesity, age-related eye diseases and cancer (1,2). Menopause is associated with an increase in oxidative stress, resulting from an imbalance between reactive oxygen species (ROS) and the antioxidant system (3–5). Previous studies have demonstrated that oxidative stress is implicated in the pathogenesis of various chronic diseases (6–9). Osteoporosis is a skeletal disorder characterized by compromised bone strength, predisposing patients to increased risk of fracture (10). Although osteoporosis is a multifactorial disorder, estrogen deficiency following menopause serves a critical role in the development of osteoporosis in women (11). There are multiple mechanisms underlying the rapid resorption of bone and loss of bone density due to estrogen deficiency, including direct effects on osteoblastic and osteoclastic cell lineages and the interaction of systemic hormones, local cytokines [including tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6], growth factors, and transcription factors (11,12). Additionally, increased ROS may also decrease bone mineral density (BMD) by inducing TNF-α expression (13).

Hormone replacement therapy (HRT) is established to prevent bone loss following menopause (14,15). Previous results have indeed demonstrated that HRT reduces the risk of fractures, even among women with low fracture risk (16). However, findings from Women's Health Initiative trials in 2002 indicated that the risks associated with HRT outweighed the benefits (17,18). Consequently, various groups have recommended limiting the use of HRT in postmenopausal women at risk of facture and women younger than 60 years or within 10 years of menopause (19). Alternatives to estrogen for the treatment of postmenopausal osteoporosis as recommended by the U.S. Food and Drug Administration (FDA) include bisphosphonates, raloxifene, calcitonin and denosumab; however, these drugs have been reported to exert drug-specific adverse effects (20). In turn, lifestyle modifications have been undertaken, such as changes in exercise and diet. A number of epidemiological studies have identified that higher fruit and vegetable intake was associated with higher BMD (21–25) and a lower fracture risk (26) in postmenopausal women. While the mechanisms underlying these bone-protective effects are yet to be fully elucidated, antioxidative nutrients and phytochemicals, including vitamin C, carotenoids and polyphenols, which are contained in fruits and vegetables, may improve bone health by scavenging ROS (27).

Anthocyanins are a class of natural polyphenol compounds responsible for the colors of flowers and fruits (28). The positive health effects of foods rich in anthocyanins include CVD prevention and anticancer, anti-inflammatory, antioxidative, anti-obesity, anti-diabetic and neuroprotective activities (29,30). Additionally, Welch et al (31) suggested an anti-osteoporotic effect of anthocyanins in a cohort study of twins, in which the differences between the highest and lowest fifths of anthocyanin intake were associated with a 3.4% higher BMD at the spine and a 3.1% higher BMD at the hip. Berries contain abundant anthocyanins and have been recognized as valuable sources of natural medicines and dietary supplements (29,32). Various studies have demonstrated that berry intake may increase antioxidant status and reduce inflammatory biomarker levels in vivo (29). Based on these findings, our group hypothesized that berry consumption may be helpful in alleviating bone resorption and bone density loss following menopause. However, a limited number of studies have investigated the impact of berry intake on bone metabolism in animals and humans (29,32). Additionally, berries are a natural source of not only anthocyanins but also vitamin C, a potent antioxidant that may also have a positive effect on bone (33,34), making it difficult to determine whether the antioxidant effects of berries are due to anthocyanins, vitamin C or both. Therefore, in the present study, the effects of an anthocyanin-rich bilberry (Vaccinium myrtillus) extract (VME) on bone metabolism were investigated in ovariectomized (Ovx) rats. Bilberry, a member of the Ericaceous family, is a low-growing shrub native to Europe and North America and is related to, while distinct from, varieties of the North American blueberry (V. corymbosum) (35). The anthocyanin-rich VME has been utilized in the treatment of various eye conditions, including cataracts and glaucoma, as well as for enhancing night vision, due to its proposed anti-inflammatory and antioxidant effects (35,36). The Ovx rat model is the current FDA-approved model for the investigation of menopausal bone changes (37). Ovx has also been demonstrated to induce oxidative stress and impair antioxidant systems in rat bone (38).

Estrogen deficiency is associated with an imbalance in bone metabolism, involving a net increase in bone resorption over formation, leading to excessive and sustained bone loss (11). The increase in bone resorption is the result of increased osteoclastogenesis and decreased osteoclast apoptosis (11,12). ROS may promote osteoclast resorption directly, by stimulating signaling associated with osteoclast differentiation and receptor activator of nuclear factor (NF)-κB (RANK), or indirectly, by stimulating osteoblast/osteoclast coupling and subsequent osteoclast differentiation through RANK ligand (RANKL) (32). A number of studies have demonstrated that berry extracts may reduce oxidative stress (45–47). Karlsen et al (48) demonstrated that anthocyanins isolated from bilberries and black currants efficiently suppressed LPS-induced activation of NF-κB in cultured monocytes. Tanabe et al (49) reported that cranberry extract inhibited RANKL-dependent differentiation of human pre-osteoclasts and bone resorption activity of osteoclasts. Furthermore, a recent study by Moriwaki et al (50) demonstrated that anthocyanin compounds extracted from bilberry and black currant inhibited osteoclast formation from osteoclast precursor RAW264.7 cells. Collectively these findings indicate anthocyanin extracted from berry fruits may alleviate bone resorption and bone density loss following menopause in women. In the present study, an anthocyanin-rich bilberry extract was administered to Ovx rats (Ovx+VME group) for 8 weeks, and BMD was measured using DXA and pQCT. In the VME-treated rats, there were no significant changes in BMD or BMC, even in trabecular or cortical BMD at the metaphyseal and diaphyseal sites, compared with those in rats in the Ovx group. These results were supported by the bone histomorphometry results (BV/TV, Tb.Th and Tb.N). Although these parameters remained stable when the rate of bone resorption was equal to that of bone formation, bone histomorphometry also revealed that the anthocyanin-rich bilberry extract did not affect bone formation (OV/BV, OS/BS, dLS/BS, MS/BS and BFR/BS) or resorption (Oc.S/BS and ES/BS). These results suggested that the anthocyanin-rich bilberry extract may not mitigate the bone losses observed in postmenopausal women.

There have been a limited number of studies that have investigated the effects of consuming berry fruits or their extracts on bone metabolism in Ovx-induced bone loss animal models. Devareddy et al (51) demonstrated that Ovx rats (6 months old) fed a diet supplemented with blueberry powder (5% w/w) for 4 months had a higher overall BMD of the whole body but not at the tibia, femur or fourth lumbar vertebra compared with rats in an Ovx group. Additionally, they identified that the supplement treatment down-regulated Ovx-induced elevation of alkaline phosphatase, collagen and tartrate-resistant acid phosphatase (TRAP) gene expression, suggesting that the bone-protective effect of blueberries may be due to the suppression of bone turnover (51). More recently, Zheng et al (52) identified that Ovx mice (14 weeks old) given a diet containing 1% anthocyanin-rich blackcurrant extract for 12 weeks had significantly greater femur BMD compared with Ovx control mice. Notably, they demonstrated that the extract reduced the number of TRAP-positive osteoclast-like cells and bone resorption activity, and concluded that the extract may alleviate bone loss by suppressing osteoclastogenesis and osteoclast function.

The reasons underlying the inconsistent results between the present study and previous studies are unknown. A previous randomized prospective study designed to examine whether the consumption of freeze-dried blackberries or blueberries (45 g daily) could prevent smoking-induced bone loss in postmenopausal women demonstrated that the loss of total body BMD was significant in women who consumed blueberries but not blackberries for 9 months, despite the higher content of anthocyanins in blueberries compared with blackberries (652.2 vs. 284.1 mg per 45 g freeze-dried berry) (53). Notably, 87% (247.1 mg) of the anthocyanins in the freeze-dried blackberries was cyanidin-3-glucoside, which was a greater percentage than the 1% (6.6 mg) in the freeze-dried blueberries. These findings suggested that the profile or ingredients, and not the total amount of anthocyanins, may be responsible for the discrepancies between previous studies (53), although the active ingredients in the anthocyanins that may prevent bone loss are yet to be identified. Kaume et al (54) fed 5% and 10% (w/w) blackberry diets rich in cyanidin-3-glucoside for 100 days to Ovx rats (9 months old) and demonstrated that the 5% (but not 10%) blackberry diet prevented a loss of BMD at the tibia, femur and fourth lumbar vertebra. However, they failed to identify any significant changes in bone formation and resorption markers following diet supplementation, and thus were unable to conclude whether the results were due to the suppression of bone resorption or acceleration of bone formation. Moriwaki et al (50) demonstrated that delphinidin, one of the aglycone nuclei of anthocyanins, prevented bone loss in Ovx mice (7 weeks old). They also observed a significant decrease in osteoclast number in delphinidin-treated, soluble RANKL-induced osteoporotic mice, and assumed that delphinidin may prevent bone loss by suppressing bone resorption. Although cyanidin and delphinidin are major anthocyanidins retained in bilberry (55), the current study identified no substantial effects on bone mass, bone formation or bone resorption parameters on examination by bone histomorphometry. Further studies are required to determine which active ingredients have bone-protective effects in berry-extracted anthocyanins.

Differences in the experimental conditions may have also resulted in discrepancies with previous studies. Zhang et al (56) fed a diet supplemented with 10% freeze-dried blueberry powder to pre-pubertal rats between postnatal day 20 (PND20) and PND34, after which the diets were either continued (long-term feeding) or switched to a control casein diet (short-term feeding). Rats were then Ovx on PND60 and sacrificed 1 or 3 weeks thereafter, and bone parameters were investigated. The results indicated that the short- or long-term blueberry diet prevented Ovx-induced bone loss at the tibia; bone histomorphometry revealed that the rats fed either short- or long-term blueberry diet had a higher BV/TV, osteoblast number and BFR/BS compared with those in rats in control groups. They also observed that the bone-protective effect of the blueberry diet was exerted through the suppression of osteoblastic cell senescence associated with acute loss of myosin expression following Ovx. The results from their study were in contrast with those from previous in vitro and in vivo studies (29,32). These other studies suggested that the effects of berry fruits or their extract on bone metabolism were more likely to be exerted through the suppression of bone resorption. Although previous in vivo studies (51,52) used mature Ovx rats, the rats used in the study by Zhang et al (56) were young Ovx rats in pre-pubertal growth stage. The ages of rats and the time after Ovx are important factors that may influence bone response following Ovx (39). In the present study, 3-month-old Ovx rat models were used to induce bone loss. Although 3-month-old rats are regarded as mature, their bone growth slows but has not stopped (39). Therefore, a favorable effect of VME on bone, if any, may have been overwhelmed by the age of the rats and/or substantial changes associated with Ovx.

In conclusion, supplementation of Ovx rats with an anthocyanin-rich bilberry extract did not prevent Ovx-induced bone loss, at least under the experimental conditions of the present study. As there has been discrepancies between results from previous studies on the effects of berry fruits or their extracts on bone metabolism, further investigations are warranted to determine whether the consumption of berry fruits or anthocyanins extracted from berry fruits may be beneficial in mitigating bone loss in postmenopausal women.