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Marine Alga Sargassum Horneri Component And Bone Homeostasis: Role In Osteoporosis Prevention

Bone is a dynamic tissue that preserves skeletal size, shape, and structural integrity and to regulate mineral homeostasis. Bone homeostasis is maintained through a balance between osteoblastic bone formation and osteoclastic bone resorption. Aging and numerous pathological processes induce decrease in bone formation and increase in bone resorption, leading to osteoporosis, a devastating bone disease [1]. Osteoporosis, which is induced with decrease in bone mass, is widely recognized as a major public health problem. The most dramatic expression of the disease is represented by fractures of the proximal femur for which the number increases as the population ages [2].


Introduction
Bone is a dynamic tissue that preserves skeletal size, shape, and structural integrity and to regulate mineral homeostasis. Bone homeostasis is maintained through a balance between osteoblastic bone formation and osteoclastic bone resorption. Aging and numerous pathological processes induce decrease in bone formation and increase in bone resorption, leading to osteoporosis, a devastating bone disease [1]. Osteoporosis, which is induced with decrease in bone mass, is widely recognized as a major public health problem. The most dramatic expression of the disease is represented by fractures of the proximal femur for which the number increases as the population ages [2].
Nutritional factors may have the potential effect to prevent bone loss with increasing age. There is growing evidence that the supplementation of nutritional and food factors may have the preventive effect on bone loss that is induced in animal model of osteoporosis and in human subjects [3][4][5][6]. Functional factors in food and plants, which regulate on bone homeostasis, have been to be worthy of notice in maintaining of bone health and prevention of bone loss with increasing age [7][8][9][10][11][12][13].
The effect of marine algae on bone metabolism was not determined so far. Undaria pinnatifida, Sargassum horneri, Eisenia bicyclis, Cryptonemia scmitziana, Gelidium amansii, and Ulva pertusa Kjellman are applied as food materials. Among these marine algae, Sargassum horneri (S. horneri) was found to have a unique anabolic effect on bone components [14]. S. horneri extract has been found to have a stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption in vitro, thereby increasing bone mass. This review has been written to outline the recent advances that have been made concerning the anabolic effect of S. horneri extract on bone metabolism and will discuss the usefulness of this functional food factor in prevention of osteoporosis.

Cellular regulation of bone homeostasis
Bone homeostasis is skillfully regulated through the functions of osteoblasts and osteoclasts, which are major cells in bone tissues [16][17][18][19][20]. Osteoclasts, which develop from hematopoietic progenitors, are recruited to the site and excavate the calcified matrix. In the physiologic process of bone turnover, a resorptive stimulus firstly triggers recruitment of osteoclasts to a site on the bone surface. During the initiation phase of bone resorption, osteoblasts arising from local mesenchymal stem cells assemble at the bottom of the cavity and bone formation begins. After the resorbed lacunar pit is filled with new osteoid, osteoblasts become flatter and less active, with the final newly remodeled bone surface lined by flat lining cells. As bone formation progresses, Yamaguchi M (2013) Marine Alga Sargassum orneri Component and Bone Homeostasis: Role in Osteoporosis Prevention. Int J Food Sci Nutr Diet. 2(1), 9-14. some osteoblasts are entombed within the matrix as osteocytes but the majority dies by apoptosis. When mechanical forces are reduced, for example in weightlessness, osteocytes die by apoptosis. This event appears to act as a beacon for osteoclast recruitment and generation of a new basic multicellular unit, which in turn replaces the old bone containing dead osteocytes with new bone containing viable osteocytes. Remodeling of cancellous bone begins with the retraction of lining cells that cover the bone surface [19,20].
Bone acts as major storage site for growth factors, which are produced by osteoblasts, diffuse into newly deposited osteoid and are stored in the bone matrix including isulin-like growth factors (IGF-I and II), transforming growth factor-β1 (TGF-β1), platelet-derived growth factor (PDGF), or bone morphologic proteins (BMPs) [21,22]. These bone-derived factors, which can be liberated during subsequent periods of bone resorption, act in an autocrine, paracrine, or delayed paracrine fashion in the local microenvironment of the bone surface.
This process of bone remodeling to make bone unique among organs and tissues, and it also adds so many levels of complexity, with respect to interactions along the remodeling sequence by systemic influences (hormones), stress action on trabecular and cortical systems (physical activity/weight bearing), growth factors and cytokines produced by the bone cells which act locally on their own cell types and on the other bone cell types, or factors that come from nearby cells present in the marrow tissues.

S. horneri extract stimulates osteoblastic bone formation
Marine algae S. horneri [Sargassum horneri (Turner) C. Agardh] was seasonally gathered from the coast at Shimoda (Shizuoka Prefecture, Japan) and Miyako (Iwate Prefecture, Japan), and it was freeze-dried and powered [14]. The gathered fresh marine algae were homogenized in distilled water and are centrifuged at 5500 g in a refrigerated centrifuge for 10 min. The 5500 g supernatant fraction was pooled for freeze-drying. The powder of the watersolubilized extract extract was dissolved in ice-cold distilled water for use in the experiments. The water-solubilized extract from S. horneri was purified through the method of membrane fractionation to collect active component of various molecular weights.
The direct effect of S. horneri extract on bone formation and mineralization has been examined using MC3T3 preosteoblastic cells in vitro [15]. The preosteoblastic cells were differentiated into mineralizing osteoblasts in vitro in the presence or absence of S. horneri extract in mineralizing medium for 21 days and stained for calcium deposition with Alizarin Red-S. S. horneri extract (10 and 25 μg/ml) was found to potently enhance mineralization in MC3T3 cells [15]. The S. horneri extract (25 -100 μg/ml of medium) did not have a significant effect on cell number of MC3T3-E1 cells with culture. S. horneri extract did not appear to mediate direct toxic effects on the cultures as cells were proliferated robustly over the culture period, and were still alive and visibly attached to the plate at the end of the experiment as compared with those of control group.
Thus, S. horneri extract has been shown to stimulate osteoblastic differentiation and mineralization in vitro. TGF-β1-and BMP-2-induced activation of Smad signaling respectively plays an important role in the early commitment and differentiation of osteoblasts [22]. S. horneri extract was found to enhance BMP-2-or TGF-β1-induced Smad activation with the dose-dependency [15]. S. horneri extract-induced Smad enhancement may be an important to stimulate osteoblastic differentiation and mineralization.
The anabolic effect of S. horneri extract on bone tissues has been found. Rat femoral-metaphyseal tissues were cultured in a medium containing water-solubilized extract (25 and 50 μg/ml) obtained from U. pinnatifida, S. horneri, E. bicyclis, or C. scmitziana in vitro [14]. The bone calcium content was significantly elevated in the presence of S. horneri extract (25 and 50 μg/ml). No effect was seen in the extracts of other marine algae. In addition, water suspensions (5%) of marine alga powder were orally administered once daily for 7 days. Bone calcium content was significantly increased after the adminostration of U. pinnatifida, S. horneri, E. bicyclis, or C. scmitziana [14]. Also, bone alkaline phosphatase activity, which is an enzyme for calcification [27,28], was significantly enhanced with the administration of S. horneri or G. amansii. Thus, S. horneri extract has a unique anabolic effect on bone calcification in vitro and in vivo. This was the first time finding. The effects of S. horneri extract in increasing calcium content, alkaline phosphatase activity, and deoxyribonucleic acid (DNA) content in the femoraldiaphyseal and -metaphyseal tissues in vitro was completely abolished in the presence of cycloheximide, an inhibitor of protein synthesis [29]. DNA may be an index of cell number in bone tissues [16,21] The anabolic effect of S. horneri extract may be resulted from newly synthesized protein components.

S. horneri extract inhibits osteoclastic bone resorption
S. horneri extract has been shown to suppress osteoclastic bone resorption. To examine the effect of S. horneri extract on osteoclast formation, RAW264.7 osteoclast precursors were differentiated into mature osteoclasts by stimulation with RANKL in the presence or absence of S. horneri extract with the dose range of 5 to 100 μg/ml of medium [15]. The S. horneri extract had no effect on cell number of preosteoclasts (RAW267.4 cells) with culture. S. horneri extract (100 μg/ml) did not have a toxicity to the precursors suppressing their proliferation over 7 days of culture [15]. S. horneri extract (25 -100 μg/ml) significantly suppressed osteoclast formation induced by RANKL [15].
The NF-ҡB signal transduction pathway is essential for the generation of osteoclasts [17,18]. The effect of S. horneri extract on NF-ҡB activation by RANKL in osteoclast precursors has been examined. RAW 264.7 cells were transfected with an NF-ҡB reporter and stimulated with RANKL to induce NF-ҡB activity in the presence or absence of S. horneri extract (5 -100 μg/ml) [15]. S. horneri extract did not have a significant effect on basal NF-κB activity [15]. However, RANKL-induced increase in NF-ҡB activity was significantly blunted in the presence of S. horneri extract [15].
The effect of S. horneri on mineralization in MC3T3-E1 cells with culture for 21 days was observed at 10 μg /ml [15]. However, the effects of S. horneri on Smad activity in MC3T3-E1 cells and on NF-ҡB activity in RAW 264.7 cells with culture for 24 hours were observed at 25 μg /ml [15]. The effects of S. horneri on osteoclastogenesis in RAW 264.7 cells with culture for 6 days were also revealed at 25 μg/ml [15]. The effect of S. horneri on osteoblastogenesis and osteoclastogenesis may be observed with lower doses in the culture for longer periods.
The effects of S. horneri extract on bone resorption using femoral tissues in vitro have been examined [30]. Parathyroid hormone (PTH)-and prostagrandine E2 (PGE2) are known to induce osteoclastic bone resorption [31][32][33]. PTH-and PGE2-induced decreases in bone calcium content were completely inhibited after culture with water-solublilized extracts (10, 25, and 50 μg/ml) [30]. Also, the extract completely inhibited the PTH-or PGE2induced increase in medium glucose consumption and latic acid production by bone tissues [30]. Moreover, S. horneri extracts blocked the PTH-induced increase in acid phosphatase activity in the diaphyseal and metaphyseal tissues [30]. These findings indicate that the water-solubilized extracts of S. horneri have a direct inhibitory effect on bone resorption in tissue culture in vitro.
S. horneri extract possesses a potent Smad activation and anti-NF-ҡB activity and may have promise for development into an antiosteoporotic material capable of promoting new bone formation while simultaneously reducing bone resorption. S. horneri extract may be usefulness as a tool in the prevention of osteolysis with various pathophysiologic states. The mechanism by which S. horneri stimulates bone mass is summarized in Fig. 1. S. horneri extracts has been found to stimulate osteoblastic bone formation [14,15,29] and to suppress osteoclastic bone resorption [30]. Those active components have been found to be present in S. horneri extract obtained from various coasts in both Japan (Shimoda and Iwate, Japan) and China [34]. The active component of S. horneri extract in stimulating bone calcification has been found to be near molecular weight (MW) 3000 [15,34]. Meanwhile, the active component of S. horneri extract in inhibiting osteoclastic cell formation was less than MW 3000 and over MW 50000 [15,34]. These components were stable under heat treatment. It has been speculated that the active component in stimulating bone calcification is a chemical but not peptide, and that the component over MW 50000 in inhibiting bone resorption may be a polysaccharide. These active components obtained from the coasts of Iwate (Japan) or China showed an identical molecular weight. The active component, which stimulates osteoblastogenesis and suppresses osteoclastogenesis, was found to be present in the components less than MW 3000 of S. horneri extract using preosteoblastic cells and RAW267.4 cells in vitro [15]. The active component that stimulates osteoblastic bone formation and suppresses osteoclastic bone resorption may be identical.

Bone mass
We found the existence of 4 chemicals in S. horneri components (less than MW 3000) using the analysis with liquid chromatography mass spectrophotometry system (LCMS-IT-TOF; Shimadzu, Kyoto, Japan). These chemicals were identified as 1,3,5-tris(oxolan-

Anabolic effects of S. horneri extract on bone in vivo
The anabolic effects of S. horneri extract on bone components in the femoral tissues of young and aged rats in vivo have been demonstrated [35]. Calcium content, alkaline phosphatase activity, and DNA content in the femoral-diaphyseal and -metaphyseal tissues of young male (4-week-old) rats were significantly increased after the administration of S. horneri extract (25, 50, and 100 mg/kg) for 7 days [35]. Moreover, these bone components in the femoral-diaphyseal and -metaphyseal tissues of aged female (50-week-old) rats were significantly increased after the administration of S. horneri extract (100 mg/kg) for 14 days, suggesting a preventive effect on bone loss with increasing age [35].
The intake of S. horneri extract has a preventive effect on bone loss in a pathophysiologic state has also been shown [36]. Diabetes has been shown to induce bone loss [37][38][39]. Streptozotocin (STZ) induces decrease in insulin secretion in pancreatic cells and causes type 1 diabetes. The oral administration of S. horneri extract (100 mg/kg body weight) to STZ (60 mg/kg body weight)-diabetic rats was found to have a preventive effect on bone loss with diabetes in vivo [36]. This finding suggests that the dietary intake of S. horneri extract has a preventive effect on bone loss in the pathophysiologic state. When the femoral tissues obtained from STZ-diabetic rats were cultured in medium containing S. horneri extract solution, the femoral calcium content and alkaline phosphatase activity were significantly increased in vitro [36]. Alkaline phosphatase is related to bone calcification [27,28]. S. horneri extract has a stimulatory effect on bone for-mation [14,15,29] and an inhibitory effect on bone resorption in vitro [15,30]. Presumably, the preventive effect of S. horneri extract administration on diabetes-induced bone loss may be related to a direct action of the active component of S. horneri extract.
Interestingly, the oral administration of S. horneri extract (100 mg/kg body weight) to STZ-diabetic rats has been found to have a significant preventive effect on the decrease in body weight and the increase in serum glucose and triglyceride levels induced in the diabetic state [36]. This was a novel finding. The intake of S. horneri extract has a partial restorative effect on serum biochemical finding with diabetes in vivo.
Thus, the intake of S. horneri extract has been demonstrated to have preventive effects on bone loss, hyperglycemia, and hyperlipidemia in STZ-diabetic rats [36]. The active component of S. horneri extract in preventing bone loss induced with diabetic state may be identical to the component that prevents an elevation of serum glucose and triglyceride levels with diabetes. This remains to be elucidated.

Supplemental intake of S. horneri extract has an anabolic effect on bone metabolism in human subjects
The supplemental intake of S. horneri extract has been shown to have an anabolic effect on bone metabolism in human subjects [40]. Study has been undertaken to determine the effect of supplemental intake of the water-solubilized S. horneri extract on circulating bone metabolic markers in healthy human [40]. Thirty-six volunteers, aged 20-60 years (16 men and 20 women), were enrolled. Volunteers were divided into three groups; placebo tablet without S. horneri extract (5 men and 7 women), tablet containing S. horneri extract at 300 mg/day (6 men and 7 women) or 900 mg/day (5 men and 6 women) [40]. Placebo or S. horneri extract tablet was ingested once a day for 4 or 8 weeks [40]. The intake of dietary S. horneri extract (900 mg/ day) for 8 weeks did not have a significant alteration in other biochemical markers for the metabolic function of organs, suggesting that the intake does not have toxic effects in humans [40].
Bone-specific alkaline phosphatase [41] and γ-carboxylated osteocalcin [42] are serum bone markers of bone formation, and bone tartrate-resistant acid phosphatase (TRACP) [43] and Ntelopeptides of type I collagen [44] are markers of bone resorption. Serum bone-specific alkaline phosphatase or γ-carboxylated osteocalcin concentration was not significantly changed after the intake of S. horneri extract (300 or 900 mg/day) for 4 or 8 weeks [40]. Serum bone tartrate-resistant acid phosphatase (TRACP) activity was significantly decreased after the intake of S. horneri extract (300 or 900 mg/day) for 8 weeks [40]. Serum N-telopeptides of type I collagen concentration was significantly decreased after the intake of S. horneri extract (900 mg/day) for 8 weeks [40]. Meanwhile, serum calcium, inorganic phosphorus, and other biochemical findings were not changed after the intake of S. horneri extract (300 or 900 mg/day) for 4 or 8 weeks [40]. Thus, the prolonged intake of S. horneri extract has been found to have inhibitory effects on bone resorption in humans.
S. horneri extract has been shown to have a stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption. It has been speculated that the supplemental intake of S. horneri extract reveals suppressive effects on bone resorption, and later it exhibits stimulatory effects on bone formation in humans, thereby increasing bone mass. The supplemental intake of S. horneri extract may have a preventive effect on bone loss with increasing age and post-menopausal women.

Conclusion
Aging and numerous pathological processes induce decrease in bone formation and increase in bone resorption, leading to osteoporosis, which is induced with decrease in bone mass. Osteoporosis is widely recognized as a major public health problem. Functional food factors may have the potential effect to prevent bone loss with increasing age. There is growing evidence that the supplementation of food factors may have the preventive effect on bone loss that is induced in animal model of osteoporosis and in human subjects. Among various marine algae which are applied to food, S. horneri extract has been found to have a unique anabolic effect on bone mass using stimulating osteoblastic bone formation and depressing osteoclastic bone resorption. The intake of S. horneri extract has usefulness in prevention of bone loss with increasing age and pathologic state. This biomedical food material may play a role in maintaining of bone health and preventing of osteoporosis.

Author Contribution
Masayoshi Yamaguchi contributed to the design and conduct of the study, collection, analysis, and interpretation of data, and manuscript writing.