JOURNAL OF MEDICINAL FOOD J Med Food 17 (12) 2014, 1298–1305 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.3095

The Antiosteoporotic Effects of Cheonggukjang Containing Vitamin K2 (Menaquinone-7) in Ovariectomized Rats Wei-Jie Wu,1,* Hwa-Young Lee,2,* Geum-Hwa Lee,2 Han-Jung Chae,2 and Byung-Yong Ahn3 1

Department of Food Science & Biotechnology, Chonbuk National University, Iksan, Korea. 2 Department of Pharmacology, Institute of Cardiovascular Research, School of Medicine, Chonbuk National University, Jeonju, Korea. 3 Department of Oriental Medicine Resources, Chonbuk National University, Iksan, Korea.

ABSTRACT The effect of dietary vitamin K2 (menaquinone-7, MK-7) and cheonggukjang (CGJ) on the prevention of ovariectomy (OVX)-induced bone loss was studied in rats. Female Sprague-Dawley rats were divided into eight groups: shamoperated; OVX control; OVX treated with MK-7 at doses of 2, 4, and 8 lg/day; and OVX treated with CGJ at doses of 0.063, 0.125, and 0.250 g/day referenced to MK-7 levels at 2, 4, and 8 lg/day, respectively. After 8 weeks of treatment, the preventive effects of MK-7 and CGJ were evaluated by measuring body weights, serum levels of bone turnover markers, bone mineral content (BMC), bone mineral density (BMD), trabecular microarchitectural properties, and bone histological characteristics. Our results showed that rats treated with a high dose of MK-7 (8 lg/day) exhibited a minor inhibitory effect on OVX-induced bone loss, as indicated by a significant increase in trabecular number, as well as BMC and BMD (P < .01). Moreover, the preventive effects of MK-7 were augmented by administration of CGJ at the same MK-7 dose. In addition, the preventive effects of CGJ were shown to be dose dependent, with the highest dose (0.250 g/day) significantly (P < .01) increasing BMC and BMD by 31.8% and 47.6%, respectively. In summary, these results suggest that administration of CGJ containing abundant levels of MK-7 may be a promising approach for the treatment and prevention of osteoporosis.

KEYWORDS:  bone metabolism  cheonggukjang  osteoporosis  ovariectomy  vitamin K2

of osteocalcin, a bone matrix protein containing c-carboxyglutamic acid, which plays a key role in bone metabolism.6 However, dietary intake of vitamin K2 is not sufficient to support full carboxylation of osteocalcin and matrix Glaproteins.5 Importantly, supplementation of vitamin K2 has been reported to increase levels of c-carboxylated osteocalcin and inhibit bone loss.3,7 The importance of vitamin K2 in bone metabolism is further supported by in vitro studies showing that vitamin K2 (menaquinone-7, MK-7) has a stimulatory effect on osteoblastic bone formation coupled with inhibitory effects on osteoclastic bone resorption.8,9 Thus, supplementation with vitamin K2 may have efficacy as a therapeutic approach to osteoporosis. Menaquinone-7 contains seven isoprene units and is abundant in fermented Asian soybeans, such as natto in Japan and cheonggukjang (CGJ) in Korea.10 A previous study has shown that habitual intake of natto containing abundant levels of MK-7 was beneficial for bone health in elderly men.11 Likewise, higher levels of MK-7 resulting from natto consumption may contribute to a relatively lower risk of fractures in Japanese women.12 Bone loss in ovariectomized (OVX) rats has been shown to be prevented by diets containing natto supplemented with MK-7,13 and prolonged intake of dietary natto, including MK-7, has further preventive effects.6

INTRODUCTION

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steoporosis is a multifactorial skeletal disease that leads to an increased risk of fracture.1 In particular, postmenopausal osteoporosis resulting from estrogen deficiency is partly involved in the deterioration of bone metabolism, which may increase the risk of fractures in the spine, forearm, and hip.2 Complications resulting from osteoporosis are associated with significant medical treatment expenses; consequently, osteoporosis has become a serious public health problem.3 Thus, there is an urgent need for nutritional and pharmacological interventions capable of preventing or altering the course of osteoporosis; however, the properties and mechanisms of such therapeutics are poorly understood. There is growing evidence that vitamin K2 is involved in bone metabolism and may have beneficial effects against osteoporosis.4,5 Vitamin K2 is essential for the c-carboxylation *These authors contributed equally to this work. Manuscript received 8 November 2013. Revision accepted 30 July 2014. Address correspondence to: Byung-Yong Ahn, PhD, Department of Oriental Medicine Resources, Chonbuk National University, Iksan 570-752, Republic of Korea, E-mail: [email protected]

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The majority of previous studies evaluating vitamin K2 supplementation for the prevention of osteoporosis have utilized dietary natto, and thus, the individual effects of MK-7 (isolated from natto) and CGJ on trabecular microarchitecture in osteoporosis have not been widely investigated. In this study, we assessed the preventive effects of the individual chemical forms of MK-7 and MK-7-abundant CGJ on OVXinduced bone loss by evaluating changes in serum biochemistry data, bone mass, trabecular microarchitectural properties, and bone histological characteristics. MATERIALS AND METHODS CGJ manufacture and chemicals Soybeans purchased from a local supermarket were washed thrice with tap water and soaked for 12 h at 4C. The soybeans were then autoclaved at 121C for 30 min and cooled to 40C before inoculation at 2% (w/w) with a precultured cell suspension of Bacillus subtilis KCTC 12392BP. The as processed soybeans were allowed to ferment for 36 h at a temperature of 43C and humidity of 85%. Finally, CGJ was harvested, and the content of MK-7 (30 lg/g) was verified by high-performance liquid chromatography, according to established procedures.14 Authentic vitamin K2 (MK-7) was purchased from ChromaDex, Inc. (Santa Ana, CA, USA), dissolved in ethanol, and suspended in sterile distilled water to make serial dilutions of 1, 2, and 4 lg/mL. CGJ was also serially diluted with sterile distilled water relative to the concentration of MK-7 at 1, 2, and 4 lg/mL. Animals Twelve-week-old female Sprague-Dawley rats weighing 180–190 g were purchased from Damul Science Co. (Daejeon, Korea). Rats were housed in an environmentally controlled facility maintained at a constant temperature (25C – 2C) and humidity (55% – 5%) and with a 12-h light/12-h dark cycle. Rats were individually housed in standard cages and provided with commercial standard diets containing 1.2% calcium and 0.8% phosphorus. The protocol used in the present study was approved by the ethics committee in Chonbuk National University ( Jeonju, Korea). To minimize suffering, rats were operated under anesthesia by a mixture of ketamine (35 mg/kg) and xylazine (10 mg/kg) administrated intraperitoneally. Experimental rats were either ovariectomized (OVX) or sham operated (Sham) according to the protocol of Cui et al.15 and allocated to one of the eight groups (n = 10 in each group) as follows: Sham (group 1), OVX (group 2), MK-7-treated OVX (groups 3–5 at doses of 2, 4, and 8 lg/day, respectively), and CGJ-treated OVX (groups 6–8 at doses of 0.063, 0.125, and 0.250 g/day, referenced to the same MK-7 levels as 2, 4, and 8 lg/day, respectively). After a 7-day healing period, rats in the MK7- and CGJ-treated OVX groups were orally administered serial doses of MK-7 or CGJ suspensions once daily for 8 weeks, while Sham and OVX groups were orally treated with volume-matched vehicles before sampling. The body

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weight of each rat was measured weekly. After 8 weeks of drug administration, blood was sampled from the abdominal aorta and stored at - 70C until biochemical measurements. The left tibia of each rat was fixed with fixative and used for further histomorphometric analysis, while the right tibia was cleared of all soft tissue and placed in sterile saline for 8– 16 h at 4C before performing a micro-CT (l-CT) scan. Biochemical analysis of serum parameters Alkaline phosphatase (ALP), serum calcium, inorganic phosphorus (IP), osteocalcin, triiodothyronine (T3), thyroxine (T4), and estradiol were determined using standard laboratory techniques. The serum ALP activity was measured with an ALP kit (Roche, Mannheim, Germany) with Modular Analytics PE (Roche). The levels of total calcium and IP in serum were determined by colorimetry using commercially available test kits (Roche). Serum levels of osteocalcin were measured with a two-site immunoradiometric assay with intra- and interassay CVs of 4.0% and 6.0%, respectively.16 Serum free T3, free T4, and estradiol were measured with free T3, free T4, and Estradiol a kits (Roche), respectively.15 Bone l-CT scanning For a detailed qualitative and quantitative two-dimensional and three-dimensional (3D) evaluation, the right tibia of each rat was scanned ex vivo at the end of the drug administration period, according to a method adapted from a previous study.15 Briefly, a 6-mm l-CT scan (70 kV, 85 lA, 1000 projections per 180o, 350 ms integration time) with an isotropic resolution of 18 lm was made of the proximal tibia using an in vivo l-CT scanner (SkyScan-1076 in vivo CT-scanner; SkyScan, Kontich, Belgium). The CT scanner was calibrated, and a beam-hardening correction algorithm was applied to all scans. In this study, bone volume fraction (BV/TV), bone surface/volume ratio (BS/BV), bone surface density (BS/TV), trabecular thickness (Tb.Th), direct trabecular separation (Tb.Sp), trabecular number (Tb.N), and trabecular pattern factor (Tb.Pf) were determined using a CT analyzer (V1.11.0.0; SkyScan). Bone mass measurements by dual-energy X-ray absorptiometry The bone mineral content (BMC) and bone mineral density (BMD) of femurs were measured using a Norland pDEXA Sabre (Fort Atkinson, WI, USA) equipped with Saber Research software (v3.6). Ex vivo measurements of the right distal femur were performed on excised bones positioned on a 3 mm thick piece of cotton placed on the bottom (thickness 1 mm) of a 10-cm-diameter culture dish at a constant location on the Plexiglas table and measured by dual-energy X-ray absorptiometry (DEXA) using a special collimator (0.8 mm diameter). The scan length was 5 cm, the scan width 2 cm, and the scan speed was 10 mm/s with a resolution of 0.2 · 0.2 mm.15,17

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RESULTS Changes in body weight The body weight changes of rats in each group are shown in Figure 1. These changes in body weight indicated that all groups started with a similar mean body weight, but at the end of the study, the average body weight of the Sham group was significantly (P < .01) lower compared with the ovariectomized groups (including OVX and all drug-administered OVX groups). However, no statistical differences were observed between the OVX control group and drug-administered (MK-7 and CGJ) OVX groups. Serum biochemical analysis

FIG. 1. Changes in body weight of rats in each experimental group. Rat groups consisted of sham-operated (Sham), OVX-control (OVX), MK-7-treated OVX (MK-1, MK-2, and MK-3, doses at 2, 4, and 8 lg/day, respectively), and CGJ-treated OVX (CGJ 1, CGJ 2, and CGJ 3, doses at 0.063, 0.125, and 0.250 g/day, with referenced levels of MK-7 at 2, 4, and 8 lg/day, respectively). Color images available online at www.liebertpub.com/jmf

Bone histomorphometry Left tibiae were decalcified in a solution of 5% formic acid (in distilled water) for 1 week and then dehydrated with methanol and embedded in paraffin. Paraffin sections were deparaffinized and sectioned at 4 lm with an AO Autocut/ Jung 1150 microtome. Sections with the widest marrow cavity near the growth plate of the metaphysis of tibiae were selected for further histological processing and histomorphometric measurements. Sections were stained with hematoxylin and eosin and examined for morphology under a light microscope.18 Statistical analysis Data were analyzed using SPSS statistical software (SPSS 19.0, SPSS Science, Chicago, IL, USA). Significant differences between samples were determined by Duncan’s multiple test at the levels of P < .05 or .01.

To assess the contribution of MK-7 and CGJ toward factors involved in antiosteoporotic reactions, we performed serum biochemical analyses (Table 1). There were no significant differences in serum levels of IP, calcium, free T3, and free T4 levels among any of the groups. The OVX groups exhibited significantly elevated levels of serum osteocalcin (P < .01) compared with the Sham group, and this phenomenon was slightly suppressed by administration of MK or CGJ, even though no significantly effects were observed. Levels of estradiol were significantly decreased in OVX rats and were not affected by treatment with either MK or CGJ. No significant changes in serum ALP levels were observed between the Sham and OVX group; however, administration of both MK and CGJ decreased serum ALP levels significantly. Micro-CT evaluation Three-dimensional images of the proximal tibia showed differences in the trabecular architecture among the various treatment groups (Table 2). Analysis of the data indicated that OVX rats exhibited significant changes in the trabecular microstructural parameters. Compared with the Sham group, OVX rats had a significantly (P < .01) decreased bone volume fraction (BV/TV), bone surface density (BS/TV), and trabecular number (Tb.N) by 60.2%, 57.5%, and 60.4%, respectively. The trabecular pattern factor (Tb.Pf) and direct trabecular separation (Tb.Sp) were significantly increased

Table 1. Effect of Treatment of MK-7 And Cheonggukjang for 8 Weeks on Serum Biochemical Parameters In Ovx Rats Parameters ALP (U/L) IP (mg/dL) Ca2 + (mM) Osteocalcin (ng/mL) T3 (ng/mL) T4 (lg/dL) Estradiol (pg/mL)

Sham

OVX

MK-1

MK-2

MK-3

CGJ 1

CGJ 2

CGJ 3

145 – 24 7.20 – 1.27 1.37 – 0.029 21.14 – 5.15 0.59 – 0.08 2.78 – 0.45 35.72 – 25.64

159.09 – 37 7.07 – 0.73 1.36 – 0.04 30.58 – 3.56# 0.56 – 0.09 2.85 – 0.68 22.66 – 9.47#

139 – 19* 7.22 – 1.10 1.36 – 0.03 30.23 – 7.29 0.61 – 0.10 3.26 – 0.83 21.33 – 9.37

133 – 20* 7.35 – 0.87 1.37 – 0.04 30.92 – 7.50 0.62 – 0.07 3.23 – 0.62 17.72 – 3.56

155 – 34 7.62 – 0.67 1.36 – 0.06 26.88 – 5.90 0.69 – 0.11 3.57 – 0.67 20.80 – 8.45

124 – 18* 7.07 – 0.51 1.42 – 0.06 26.10 – 4.20 0.60 – 0.08 3.37 – 0.60 21.97 – 5.97

129 – 13* 6.58 – 0.56 1.40 – 0.07 26.52 – 2.70 0.63 – 0.06 3.02 – 0.52 20.59 – 9.03

118 – 12* 6.74 – 0.57 1.35 – 0.06 33.89 – 8.76 0.68 – 0.05 2.52 – 0.63 20.55 – 11.74

Rat groups consisted of sham-operated (Sham), OVX-control (OVX), MK-7-treated OVX (MK-1, MK-2, and MK-3, doses at 2, 4, and 8 lg/day, respectively), and CGJ-treated OVX (CGJ 1, CGJ 2, and CGJ 3, doses at 0.063, 0.125, and 0.250 g/day, with referenced levels of MK-7 at 2, 4, and 8 lg/day, respectively). *P < .05 versus OVX. # P < .05 versus Sham. Rat groups as defined above (n = 10/each group).

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Table 2. Trabecular Microstructural Properties of the Right Tibial Metaphysis and Cortical Geometric Properties Evaluated Ex Vivo Using Micro-CT Parameters BV/TV (%) BS/BV (1/mm) BS/TV (1/mm) Tb.Pf (1/mm) Tb.Th (mm) Tb.N (1/mm) Tb.Sp (mm)

Sham

OVX

MK-1

MK-2

MK-3

CGJ 1

CGJ 2

CGJ 3

29.06 – 0.34a 38.13 – 2.12 11.08 – 3.17a 5.27 – 0.42a 0.11 – 0.06 2.73 – 0.67a 0.21 – 0.05a

11.57 – 0.27cd 40.73 – 3.53 4.71 – 1.56bc 11.35 – 2.20b 0.11 – 0.02 1.08 – 0.04c 0.74 – 0.08c

10.83 – 0.93cd 42.61 – 13.48 4.61 – 0.64bc 12.81 – 1.85b 0.10 – 0.01 1.04 – 0.30c 0.82 – 0.09cd

10.24 – 3.75d 41.16 – 16.39 4.22 – 1.72c 12.25 – 2.78b 0.11 – 0.03 0.96 – 0.08c 0.92 – 0.05d

13.25 – 2.05c 41.90 – 5.39 5.55 – 2.61bc 10.83 – 3.26b 0.11 – 0.03 1.24 – 0.28bc 0.70 – 0.07c

12.03 – 1.81cd 41.49 – 0.88 4.99 – 0.53bc 12.35 – 1.48b 0.11 – 0.01 1.11 – 0.03c 0.75 – 0.08c

14.94 – 1.20bc 38.19 – 1.46 5.70 – 0.41bc 9.74 – 1.20b 0.11 – 0.01 1.37 – 0.07bc 0.74 – 0.06cd

18.00 – 0.67b 39.58 – 2.38 7.12 – 0.21b 9.57 – 2.66b 0.11 – 0.01 1.69 – 0.29b 0.49 – 0.12b

Values are mean – SD. Values that do not share the same superscript letters within a row are significantly (P < .05) different from each other; n = 10 rats per group. Rat groups consisted of sham-operated (Sham), OVX-control (OVX), MK-7-treated OVX (MK-1, MK-2, and MK-3, doses at 2, 4, and 8 lg/day, respectively), and CGJ-treated OVX (CGJ 1, CGJ 2, and CGJ 3, doses at 0.063, 0.125, and 0.250 g/day, with referenced levels of MK-7 at 2, 4, and 8 lg/day, respectively).

(P < .01), although no significant differences in bone surface/volume ratio (BS/BV) and trabecular thickness (Tb.Th) were observed among any of the groups. Oral administration of CGJ exhibited a tendency toward dose-dependent preventive effects. Especially, CGJ treatment with 0.250 g/day (group of CGJ 3) significantly attenuated reduction of BV/ TV, BS/TV and Tb.N as well as prevented increases in Tb.Sp (P < .01) compared with OVX control rats. In the case of MK-7 administration, only the maximum dosage group (8 lg/day) had a significant preventive effect on BV/TV and Tb.N, while MK-7 did not exhibit any significant effects on the microarchitectural properties of the proximal tibia at lower dosages. Coronal images of the medial proximal tibiae and 3D images generated by l-CT are shown in Figure 2, which also suggested that both MK-7 and CGJ had dosedependent preventive effects on bone loss in OVX rats. BMC and density The BMC and BMD of femurs were significantly (P < .01) decreased in the OVX group compared with the Sham group (Fig. 3). Conversely, administration of MK-7 and CGJ significantly (P < .01) increased the femur BMC of OVX rats in a dose-dependent manner (Fig. 3A). Treatment with MK-7 at doses of 2, 4, and 8 lg/day increased the BMC of OVX rat femurs by 1.4%, 4.0%, and 6.4%, respectively. Meanwhile, administration of CGJ (0.063, 0.125, and 0.250 g/day, with the referenced MK-7 content at 2, 4, and 8 lg/day) increased the BMC of OVX rat femurs by 10.3%, 15.6%, and 31.8%, respectively, which was significantly higher when compared with MK-7 treatment alone. Similar patterns were also obtained for the femur BMD assay (Fig. 3B). The maximum dose of MK-7 (8 lg/day) notably increased the femur BMD by 10.6% (P < .01), which was further enhanced to 47.6% (P < .01) by administration of CGJ (0.250 g/day) at the same dose of MK-7. Histological evaluation of tibia Upon histological study of the tibiae, the cancellous bone of the Sham group showed intervening trabecular bone with connectivity of the trabecular elements, revealing normal size, shape, density, and architecture (Fig. 4). However, the

OVX group sections exhibited sparse, disrupted, spacing enlarged, and area-diminished trabecular bones compared with the Sham group. MK-7 and CGJ treatment tended to cause dose-dependent preventive effects at the experimental dosages. In particular, rats treated with a high dose of MK-7 (8 lg/day) or with CGJ, at any of the doses administered (0.063, 0.125, and 0.250 g/day), exhibited remarkable restorative progress of bony architectures. DISCUSSION In this study, the preventive effects of the chemical form of MK-7 and MK-7-rich cheonggukjang (Korean traditional fermented soybean) on bone loss were compared using an ovariectomized rat model. Ovariectomy of rats resulted in significantly increased body weight (Fig. 1), which was in agreement with previous studies.19–21 Ovariectomy-induced weight gain can be reversed by estrogen replacement and can also be prevented by administrating with soybean proteins or isoflavones in rats.19,20,22 However, in this study, drug administration did not affect the body weight growth patterns, suggesting that the action mechanisms of MK-7 and CGJ are different from estrogen-like compounds. Moreover, the serum osteocalcin patterns observed during CGJ administration in this study (Table 1) were also different from previously reported patterns by feeding estrogen.23 It should be emphasized that CGJ is a fermentation product of soybeans. Importantly, in our study, administration of a small amount of CGJ (maximum dose of 0.250 g/ day) did not contribute to this phenomenon, suggesting that any estrogen-like effects from the soybean composition of treatments could be ignored. Based on serum biochemical assessment, OVX did not affect the levels of serum IP, calcium, free T3, or free T4 levels (Table 1). Calcium is an essential structural component of the skeleton, and its metabolism plays an important role in bone turnover. Likewise, IP, T3, and T4 may contribute to the maintenance of calcium homeostasis.24 The levels of serum phosphorus and calcium did not change among any of the groups, suggesting that homeostatic mechanisms were able to maintain serum levels of these minerals despite ovariectomy and also that MK-7 and CGJ

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FIG. 2. Image analysis of medial proximal tibiae of rats from different experimental groups. (A) Coronal images of rat medial proximal tibia taken by l-CT. (B) Three-dimensional images taken by l-CT. Note: Rat groups consisted of sham-operated (Sham), OVX-control (OVX), MK-7-treated OVX (MK-1, MK-2, and MK-3, doses at 2, 4, and 8 lg/day, respectively), and CGJ-treated OVX (CGJ 1, CGJ 2, and CGJ 3, doses at 0.063, 0.125, and 0.250 g/day, with referenced levels of MK-7 at 2, 4, and 8 lg/day, respectively).

had no influence on these parameters. Osteocalcin is a noncollagenous protein secreted by osteoblasts that participates in mineralization and calcium ion homeostasis; therefore, it can be implicated as an important indicator of bone formation.21 Osteocalcin level increases during the process of bone degradation.25 The serum osteocalcin levels in rats from the OVX group were significantly higher than those in the Sham group (Table 1), and this phenomenon was not significantly affected by administration of MK-7 or CGJ. As reported, serum osteocalcin is actively involved in both bone formation and resorption.26 However, the effects of antiosteoporotic materials on osteocalcin regulation were not consistent in previous studies. In some studies, they had demonstrated that CGJ and other antiosteoporotic materials reduced serum osteocalcin levels in OVX rats, which in turn

decreased the rate of bone metabolism.21,25,27 Some other studies indicated that feeding of genistein (a soybeanderived isoflavone) increased the serum osteocalcin level and the bone formation in OVX rats.26,28 In contrast to these studies, other researchers also reported that materials with antiosteoporotic effects had no influence on serum osteocalcin,15,18 which are similar to the present study. Therefore, additional evidence is needed to further ascertain the regulatory function of MK-7 and CGJ on bone metabolism with respect to osteocalcin. Total serum ALP is a widely applied marker of bone ossification, because its level increases in the case of bone disease as well as during rapid bone growth after a fracture or osteoporosis.25 In this study, serum ALP levels were slightly increased by OVX compared with the Sham group,

ANTIOSTEOPOROTIC EFFECTS OF CHEONGGUKJANG

FIG. 3. Effect of MK-7 and cheonggukjang on bone mineral content (BMC, A) and bone mineral density (BMD, B) in OVX rats. Result are expressed as the mean – SD; bars that do not share the same superscript letters are significantly (P < .01) different from each other; n = 10 rats per group. Rat groups consisted of sham-operated (Sham), OVX-control (OVX), MK-7-treated OVX (MK-1, MK-2, and MK-3, doses at 2, 4, and 8 lg/day, respectively), and CGJ-treated OVX (CGJ 1, CGJ 2, and CGJ 3, doses at 0.063, 0.125, and 0.250 g/day, with referenced levels of MK-7 at 2, 4, and 8 lg/day, respectively).

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although no statistically significant difference was observed (Table 1). Similar to other antiosteoporotic materials,27,29 the increase of ALP was significantly inhibited by administration of either MK-7 or MK-7-abundant CGJ. However, the inhibitory effect of MK-7 and CGJ in the current study was highly effective and resulted in lower-than-normal levels of ALP (Table 1). We are presently unable to offer a reasonable explanation for this phenomenon, as serum ALP is primarily sourced from the liver and osteoblasts.30 Considering that MK-7 can stimulate the ALP activity in osteoblastic cells,9 we suspect that the lower-than-normal levels of ALP induced by administration of MK-7 and MK-7abundant CGJ were likely associated with the liver function of OVX rats. Moreover, previous studies demonstrated that the turnover of vitamin K in the bone is slower than in the liver,31 which may be the reason for the abnormal phenomenon of ALP levels as obtained in this study. Similar to a previous report,15 estradiol was significantly decreased in OVX rats, the reduction of which was not restored by MK-7 or CGJ (Table 1). The estrogen deficiency induced by OVX leads to a greater increase in the rate of bone turnover and more rapid loss of bone mass of the trabecular bone.32 In agreement with the results of previous studies, histological analysis revealed sparse, disrupted trabecular bone in OVX rats. Likewise, disruption of the trabecular network has been reported to decrease the compressive bone strength of the femur.18 In the present study, restoration of the trabecular network with less intertrabecular spaces was observed in CGJ treatment groups and the high dose of MK-7 (8 lg/day) administration group (Fig. 4). BMC and BMD were measured as surrogate markers of antiosteoporotic effects (Fig. 3). Our results indicated that the BMC and BMD of femurs were significantly (P < .01) reduced by OVX, and that these decreases were remarkably prevented by administration of MK-7 or CGJ in a dose-dependent manner. Interestingly, at the same maximum dose of MK-7 level (8 lg/day), the preventive effects of MK-7 were significantly potentiated in CGJ (31.8% for BMC and 47.6% for BMD) compared with

FIG. 4. Histochemical section images of proximal metaphysis of rat tibiae. T: trabecular bone; M: marrow spaces. (HE, 100 · ). Rat groups consisted of sham-operated (Sham), OVXcontrol (OVX), MK-7-treated OVX (MK-1, MK-2, and MK-3, doses at 2, 4, and 8 lg/day, respectively), and CGJ-treated OVX (CGJ 1, CGJ 2, and CGJ 3, doses at 0.063, 0.125, and 0.250 g/day, with referenced levels of MK-7 at 2, 4, and 8 lg/day, respectively). Color images available online at www.liebertpub.com/jmf

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the chemical form of MK-7 alone (6.4% for BMC and 10.6% for BMD). Our results are consistent with previous studies that showed consumption of CGJ alleviates OVXinduced osteoporosis by significantly increasing BMC and BMD in rats.25,27 BMC and BMD have been suggested to serve only as surrogate measures of bone strength,20 and thus, additional parameters such as trabecular microstructural properties are necessary to evaluate the true impact of a treatment on trabecular bone quality. Consistent with our results shown above, OVX induced substantial trabecular bone microstructure deterioration in the proximal metaphysis of the tibia, which manifested as reduced BV/TV, BS/TV, and Tb.N, and improved Tb.Pf and Tb.Sp (Table 2). Restoration of Tb.N is an important step toward improving bone strength, and our results showed that treatment with MK-7 at a high dosage (8 lg/day) was able to slightly inhibit the detrimental effects of OVX in the tibia. As such, the increase in Tb.N may have been responsible for the decrease in Tb.Sp.20 Moreover, the preventive effect was further enhanced by the administration of CGJ at a comparatively lower dosage (0.125 g/day), which was most significantly prevented by CGJ at a comparatively higher dosage (0.250 g/day). Based on these results, we assumed that MK-7 prevented OVX-induced decreases in bone components in a dose-dependent manner. In addition, administration of CGJ at the same level of MK-7 resulted in a significantly higher preventive efficacy compared with the chemical form of MK-7 alone, which was further confirmed by coronal images of the medial proximal tibiae and 3D images (Fig. 2). Data shown in this study indicated that MK-7 at a high dose had a slight antiosteoporotic effect, while CGJ at medium and high dosages had a more potent effect. It appears that MK-7 in CGJ played only a minor role in the antiosteoporotic effect of CGJ, and the component(s) in CGJ that played a major or synergic role in the antiosteoporotic effect still remained unclear. However, MK-7 has been proven to stimulate osteoblastogenesis and suppress osteoclastogenesis simultaneously in in vitro studies.8,9,33 In addition, feeding natto (a similar product to CGJ) diets, including MK-7, significantly prevented bone loss, while such an effect was not observed by feeding natto without MK-7, which indicates that MK-7 contained in natto diets has a significant role in the prevention of OVX-induced bone loss.13 Therefore, we speculated that the lower preventive efficacy of the chemical form of MK-7 might have been due to its poor absorption. Specifically, MK-7 is a liposoluble vitamin and its administration in the form of a suspension may have reduced its absorptivity, possibly resulting in decreased efficacy. However, in fermented soybean (CGJ), other bioactive materials also generated during fermentation, which may have enhanced the absorption rate and efficacy of MK-7. For example, poly-c-glutamic acid is generated during soybean fermentation34 and has shown great potential as a drug delivery platform.35 Moreover, other substances generated during soybean fermentation have also been reported to increase the calcium content of bones and prevent osteoporosis25,36 and, thus, may have

further augmented MK-7 to enhance the preventive efficacy on bone loss. Further studies are needed to define the association between these bioactive materials and MK-7. In conclusion, the results of the present study provide further support for use of dietary MK-7 for preventing bone loss, as evidenced by slightly improved bone mass and restoration of the trabecular network of the proximal tibia in OVX rats. Administration of CGJ containing MK-7 resulted in a significantly higher preventive efficacy compared with treatment of MK-7 alone, indicating that CGJ diets containing MK-7 may be potentially useful for the prevention of osteoporosis.

ACKNOWLEDGMENTS This research was financially supported by the Ministry of Trade, Industry & Energy (MOTIE), Korea Institute for Advancement of Technology (KIAT), and Honam Institute for Regional Program Evaluation through the Leading Industry Development for the Economic Region. AUTHOR DISCLOSURE STATEMENT No competing financial interests exist. REFERENCES 1. Klibanski A, Adams-Campbell L, Bassford T, et al.: Osteoporosis prevention, diagnosis, and therapy. JAMA 2001;285:785– 795. 2. Ushiroyama T, Ikeda A, Ueki M: Effect of continuous combined therapy with vitamin K2 and vitamin D3 on bone mineral density and coagulofibrinolysis function in postmenopausal women. Maturitas 2002;41:211–221. 3. Tsukamoto Y: Studies on action of menaquinone-7 in regulation of bone metabolism and its preventive role of osteoporosis. Biofactors 2004;22:5–19. 4. Takeuchi Y, Suzawa M, Fukumoto S, Fujita T: Vitamin K2 inhibits adipogenesis, osteoclastogenesis, and ODF/RANK ligand expression in murine bone marrow cell cultures. Bone 2000; 27:769–776. 5. Knapen MHJ, Drummen NE, Smit E, Vermeer C, Theuwissen E: Three-year low-dose menaquinone-7 supplementation helps decrease bone loss in healthy postmenopausal women. Osteoporos Int 2013;24:1–9. 6. Yamaguchi M, Kakuda H, Gao YH, Tsukamoto Y: Prolonged intake of fermented soybean (natto) diets containing vitamin K2 (menaquinone-7) prevents bone loss in ovariectomized rats. J Bone Miner Metab 2000;18:71–76. 7. Tsukamoto Y, Ichise H, Kakuda H, Yamaguchi M: Intake of fermented soybean (natto) increases circulating vitamin K2 (menaquinone-7) and c-carboxylated osteocalcin concentration in normal individuals. J Bone Miner Metab 2000;18:216–222. 8. Yamaguchi M, Ma Z: Inhibitory effect of menaquinone-7 (vitamin K2) on osteoclast-like cell formation and osteoclastic bone resorption in rat bone tissues in vitro. Mol Cell Biochem 2001; 228:39–47. 9. Yamaguchi M, Sugimoto E, Hachiya S: Stimulatory effect of menaquinone-7 (vitamim K2) on osteoblastic bone formation in vitro. Mol Cell Biochem 2001;223:131–137.

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The antiosteoporotic effects of Cheonggukjang containing vitamin k2 (menaquinone-7) in ovariectomized rats.

The effect of dietary vitamin K2 (menaquinone-7, MK-7) and cheonggukjang (CGJ) on the prevention of ovariectomy (OVX)-induced bone loss was studied in...
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