ARTICLE pubs.acs.org/JAFC
Kudzu (Pueraria lobata) Vine Ethanol Extracts Improve Ovariectomy-Induced Bone Loss in Female Mice Teruyoshi Tanaka, Hanjun Tang, Fengnian Yu, Seiwa Michihara, Yuki Uzawa, Nobuhiro Zaima, Tatsuya Moriyama, and Yukio Kawamura* Department of Applied Biological Chemistry, Graduate School of Agriculture, Kinki University, 3327-204, Naka-machi, Nara 631-8505, Japan ABSTRACT: Bone-loss-improving action of kudzu vine ethanol extracts (PVEE) was clarified. PVEE was composed roughly of 80% fiber, 10% puerarin, 3.6% daidzin, 2.5% 600 -O-malonyldaidzin, and the other minor isoflavones. Ten-week-old ovariectomized (OVX) mice were fed diets containing PVEE (20 mg/kg body weight/day) for 8 weeks. The bone resorption markers (urinary deoxypyridinoline and tartrate-resistant acid phosphatase activity) was elevated in OVX mice and was significantly decreased in OVX mice that consumed PVEE for 8 weeks. Consistent with the decrease in the markers, the number of matured osteoclasts in the distal femur was diminished in OVX mice fed PVEE diets. PVEE diets also suppressed the decrease in femoral bone mineral density (BMD) by OVX. PVEE showed the affinity for estrogen receptor α and β nearly 1/10000 weaker than 17β-estradiol. No hypertrophy in the uterus by the PVEE diet was observed. These results suggest that PVEE could be a promising resource for a functional food that improves osteoporosis. KEYWORDS: Pueraria lobata, isoflavone, puerarin, osteoporosis, bone resorption
’ INTRODUCTION Bones weaken with age, and decreased bone density becomes physiologically apparent at approximately 45 years old or a later age when the risk of osteoporosis increases in most women. Decreased bone density and the resulting bone dysfunction greatly affect the quality of life because the bone is a supporting organ, hematopoietic tissue, and immune tissue. In recent years, the number of osteoporosis patients has increased as the aging population has grown, and bone health is becoming a serious problem. Bone health is profoundly affected not only by exercise but also by nutrition.1 Calcium2 and vitamin D3 have been extensively studied as preventive nutrients for osteoporosis. Recently, vitamin K was reported to play a role in enhancing bone mineralization through the γ-carboxylation of osteocalcin.4,5 Therefore, consuming fruits and vegetables rich in minerals and vitamins helps maintain bone health.1 Flavonoids and carotenoids are also abundant in fruits and vegetables. Soybeans and soybean isoflavones have been well examined for their effects on bone metabolism.69 However, little is known about effects of other isoflavone-rich food sources on bone metabolism. Kudzu (Pueraria lobata) is a creeping and climbing plant with long vines of the Leguminosae family that has spread worldwide and is predominant in temperate climates. Puerariae radix, often called “kakkon”, is the dried root of kudzu. Kakkon has traditionally been used as a herbal medicine to treat common cold, headaches, diarrhea, and hypertension in Oriental countries including Japan. It was recently demonstrated that dietary kakkon isoflavone lowered arterial pressure in stroke-prone spontaneously hypertensive rats.10 Wang et al. reported that Pueraria radix prevented bone loss in ovariectomized mice.11 However, in Japan the Pharmaceutical Affairs Law does not allow the kudzu root as a resource for food except for starch. Thus, we examined whether the other parts have any potential for bone r 2011 American Chemical Society
health. To our knowledge, there are no reports on the effects of kudzu vine extracts on bone metabolism. We previously reported that Kudzu (Pueraria lobata) vine ethanol extracts (PVEE) suppressed the differentiation of RAW264.7 cells, a preosteoclastic cell line, by downregulating NFATc1, a key transcription down regulating NFATc1 a transcriptionfactor for osteoclastspecific genes.12 In the present study, we investigated whether the PVEE diet to ovariectomized (OVX) mice prevented the bone loss by ovariectomy and provide a novel finding that a realistic doze of PVEE diet to OVX mice improves postmenopausal bone loss by downregulating the differentiation of the osteoclast in the bone marrow.
’ MATERIALS AND METHODS PVEE. Kudzu vines were collected on the campus of Kinki University, School of Agriculture, and extracted at the Nara Prefectural Institute of Industrial Technology. Briefly, threefive volumes of ethanol were added to the kudzu vines based on the weight of the collected kudzu and then homogenized. After filtration, the extracts were concentrated with an evaporator. Finally, the extracts were frozen in liquid nitrogen and dried. The isoflavone content of PVEE was quantitated by HPLC using a Cosmosil 5C18-AR-II 250 4.6 mm column (Nacalai Tesque, Kyoto, Japan) with a gradient from solution A, a 10% acetonitrile aqueous solution (0.1% formic acid), to solution B, a 35% acetonitrile aqueous solution (0.1% formic acid), at a flow rate of 1 mL/min. Peaks were detected at 260 nm. Animals and Diets. Slc:ddY female mice were purchased from Japan SLC (Shizuoka, Japan). They had either been ovariectomized Received: August 5, 2011 Accepted: November 7, 2011 Revised: November 1, 2011 Published: November 07, 2011 13230
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Journal of Agricultural and Food Chemistry (OVX) or sham-operated (Sham) at 9 weeks of age. Sham and OVX mice were divided into the following 4 groups: Sham-control group, Sham-PVEE group, OVX-control group, and OVX-PVEE group. The mice were housed in individual cages under a 12 h/12 h light/dark cycle at 20 ( 2 °C. A commercial diet (MF, Oriental Yeast, Tokyo, Japan) was used as a control diet. The PVEE diet (isoflavone content: 20 mg/kg body weight/day, approximately 0.66 mg isoflavones/day/mouse in 4.5 g diet) was made by mixing PVEE with MF powder. This dosage was calculated from the daily isoflavone allowance [3040 mg supplementation/day/person (60 kg)] recommended in Japan, which corresponded to 0.5 to 0.66 mg/day/mouse. The efficacy of this dosage was preliminarily ascertained by the elevation of urinary DPD/Cre after the administration to 5 OVX mice for 2 weeks (data not shown). Because a very small amount of PVEE was added to the fundamental diet, the compensation by additional cornstarch was not performed. The diet and tap water were supplied ad libitum. During the experimental period, body weight and food intake were measured once a week. After administering the diet for 8 weeks, the mice were sacrificed, and the heart, liver, spleen, kidney, and uterus were harvested and weighed. The right humerus was isolated to quantitate the gene expression level of tartrate-resistant acid phosphatase (TRAP) in the bone marrow cells. The left femur was used to determine the TRAP activity in the bone marrow cells. The right femurs were collected and then ground to analyze the bone microstructure. The distal area of the left femur was TRAP-stained. All animals were treated in accordance with rules for animal experiments at Kinki University and International Guiding Principles for Biomedical Research Involving Animals (1985). Collection of Urine and Blood Samples. Urine and blood samples were collected at 0, 4, and 8 weeks. Urine was collected directly from the bladder. Blood samples were taken from the tail vein and then centrifuged to collect the serum. Both urine and blood samples were stored at 80 °C until further analysis.
Extraction of Total RNA from Humeri and Quantitative RTPCR. Total RNA was isolated from the right humeri by using the RNAqueous kit (Ambion, Austin, TX, USA). The RNA was subsequently treated with a TURBO DNase-free kit (Ambion) to remove genomic DNA contamination. Two micrograms of each sample was reverse transcribed into cDNA with the high capacity cDNA reverse transcription kit (Applied Biosystems). For a quantitative analysis of each gene expression, the amplification of cDNAs was performed using SYBR Premix Ex Taq (Takara, Shiga, Japan), primers [TRAP: forward primer, 50 -GGAAATGGCCAATGCCAAAG-30 ; reverse primer, 50 -ATCATGGTTTCCAGCCAGCAC-30 . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward primer, 50 -ATGGTGAAGGTCGGTGTGAA-30 ; reverse primer, 50 GAGTGGAGTCATACTGGAAC-30 ] with a Thermal Cycler Dice (Takara) according to the manufacturer’s protocol. The expression data of the TRAP gene was normalized to the GAPDH expression level of the same individual sample. Protein Extraction from Bone Marrow Cells. Both ends of the excised left femurs of mice were cut, and the bone marrow cells were flushed with saline. The collected bone marrow cells were recovered by centrifugation (1500g, 10 min, 4 °C) and then resuspended with saline and centrifuged (1800g, 3 min, 4 °C). This washing procedure was repeated three times. Collected samples were resuspended in 200 μL of lysis buffer (pH 7.2, 10 mM HEPES, 1 mM EDTA, 10 mM EGTA, 0.05% Triton X-100, 10% sucrose, 1 mM DTT, 1 mM AEBSF, and 0.1 mM ALLN), sonicated (Sonifier 450, Branson, CT, USA) for 30 s, and then centrifuged (8000g, 5 min, 4 °C). The supernatants were collected as the protein extracts of bone marrow cells and stored at 80 °C until further analysis. Examination of Bone Resorption Markers. The deoxypyridinoline (DPD) level in urine was measured using a commercial kit (DS Pharma Biomedical, Osaka, Japan). Urinary creatinine was measured by the picric acid method (Commercial kit from R&D Systems, MN, USA).
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The DPD levels are expressed as the ratio of urinary DPD to the creatinine (Cre) levels (DPD/Cre, nM/mM 3 Cre). TRAP activity in both the serum and protein extracts of bone marrow cells was measured by the colorimetric method. Briefly, 10 μL of serum or bone marrow cell extracts was added to 80 μL of citric acid buffer containing para-nitrophenylphosphoric acid disodium salt substrates and incubated for 60 min at 37 °C. The reaction was stopped by adding 160 μL of 0.1 N NaOH, and the absorbance at 405 nm was measured with a spectrophotometer (U-0080D, HITACHI, Tokyo, Japan). The TRAP activity in bone marrow cells was normalized to the bone marrow protein level (U/μg protein). The protein levels in each sample were determined by the Bradford assay using a protein assay kit (Bio-Rad Laboratories, CA, USA). One unit is defined as the activity that hydrolyzes 1 μmol of substrate per minute at 37 °C. Examination of a Bone Formation Marker. Serum bonespecific alkaline phosphatase (BAP) activity was measured by the colorimetric method in the presence of L-phenylalanine (inhibitor of intestinal alkaline phosphatase) as reported by Dimai et al.13 The absorbance at 405 nm was measured with a spectrophotometer (U-0080D). Histomorphometric Analysis. The femoral distal area was collected and stained with a TRAP Staining kit (Primary Cell, Hokkaido, Japan), embedded in Tissue-Tek (Sakura Finetechnical, Tokyo, Japan), and quickly frozen in liquid nitrogen. The samples were stored at 80 °C until further analysis. Subsequently, the tissues were sectioned with a cryostat and stained with Harris’s hematoxylin and then examined by light microscopy with a BX40 (Olympus, Tokyo, Japan). CT Analysis. Both the right and left femurs were scanned with an X-ray CT system (LCT-100, ALOCA, Tokyo, Japan). Tomograms were taken at 0.2-mm intervals. The bone density (cortical bone density, cancellous bone density, and total bone density) of each femur was measured. The bone density is expressed as the mean of the right and left bone densities. 3D Imaging of the Spine. The spines of mice were scanned at 0.2mm intervals with an X-ray CT system, and tomograms were constructed with 3D image software (VGStudio MAX1.2, Nihon Visual Science, Tokyo, Japan). Cross-sections of the constructed images were compared. Stereoscopic Observation of Femoral Trabeculae. The distal portions of the isolated right femur were ground with a whetstone. The produced pulverized preparation was observed with a stereoscopic microscope (SZ61, Olympus, Tokyo, Japan).
Determination of the Binding of PVEE to Estrogen Receptors. The binding of PVEE to the estrogen receptors (ERs) was determined using a commercial ER-binding kit (EnBio RCAS, EnBioTec Laboratories, Tokyo, Japan). Statistical Analysis. Data are presented as the means ( standard deviation (SD). One-way analysis of variance followed by a post-hoc Tukey test was used to examine differences between the different groups. P < 0.05 was considered statistically significant.
’ RESULTS Isoflavone Composition of PVEE. Feeze-dried and desiccated PVEE contains nearly 20% isoflavones, 7980% carbohydrate (mostly fiber), and a small amount of ashes and water. Figure 1 shows the HPLC chromatogram of isoflavones in PVEE when 10 μL of PVEE (5 mg/L) was injected into the HPLC system. Isoflavone contents of PVEE were quantitated, based on the authentic isoflavone standard as shown in Table 1. Of the isoflavones in PVEE, the most abundant was puerarin (nearly 50%) followed by daizin (3.58%), 600 -O-malonyldaidzin (2.52%), 600 -O-acetydaidzin (1.27%), and daidzein (0.92%). The chemical structures of puerarin, daidzin, and daidzein are shown in 13231
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Table 1. Isoflavones in PVEE isoflavone puerarin
Figure 1. HPLC chromatogram of isoflavones in PVEE at UV 260 nm. Peak identification: (1) puerarin, (2) daidzin, (3) glycitin, (4) 600 -Omalonyldaidzin, (5) 600 -O-malonylglycitin, (6) 600 -O-acetylglycitin, (7) genistin, (8) 600 -O-acetyldaidzin, (9) 600 -O-malonylgenistin, (10) daidzin, (11) glycytein, (12) 600 -O-acetylgenistin, (13) genistein, and (14) formononetin.
Figure 2. The other isoflavones were below 0.5%. Genistein content was as low as 0.03%. Another marked characteristic of the isoflavones in PVEE was that 98.98% was of the glycoside form, and only 1.02% was of the aglycon form. Effects of the PVEE Diet on Body Weight and Food Intake. The effects of the PVEE diet on bone metabolism were investigated in OVX mice. Ten-week-old Sham-PVEE group (n = 6) and OVX-PVEE group (n = 6) were fed PVEE-containing diets (isoflavone content: 20 mg/kg body weight/day) for 8 weeks. The body weight and food intake were measured once a week, and the changes during the experimental period are shown in Figure 3. The OVX-control group gained significantly more weight than the Sham-control group throughout the study period. PVEE feeding reduced this weight gain in OVX mice, while there were no changes in the Sham mice. Since food intake did not change in any of the groups, these data suggest that PVEE improved obesity without causing hypophagia in OVX mice, possibly due to improving lipid metabolism. This consideration was supported by the CT-imaging of abdominal fat (data not shown). Effects of the PVEE Diet on Gene Expression of TRAP in the Bone Marrow in Mice. At first, we quantitated the gene expression levels of TRAP, a specific marker of mature osteoclasts, to know the effects of PVEE on osteoclast differentiation. Figure 4 shows the TRAP gene expression level in mice fed the PVEE diet. The expression of TRAP in bone marrow in the OVX-control group markedly increased compared to that of the Sham-control group. Dietary PVEE significantly lowered the elevated expression of TRAP in OVX mice, suggesting the positive action of PVEE diet on bone absorption. Effects of the PVEE Diet on Bone Metabolic Markers. To determine the effects of PVEE on the bone metabolic state in mice, four bone metabolic markers were measured: the urinary DPD levels as a bone absorption marker, the serum and bone marrow TRAP activity as differentiation indices of osteoclast, and the serum BAP activity as a bone formation marker. As shown in Figure 3, the urinary DPD levels of the OVX-control group were consistently elevated compared to that in the Sham-control group. When OVX mice were fed the PVEE diet for 8 weeks, the urinary DPD levels significantly declined (Figure 5A). OVX treatment increased the serum TRAP activity at 4 weeks and 8 weeks (Figure 5B). The serum TRAP activity in the OVX-PVEE
content (g/100 g) 10.12
daidzin
3.58
glycitin
0.08
600 -O-malonyldaidzin
2.52
600 -O-malonylglycitin
0.00
600 -O-acetylglycitin
0.49
genistin
0.28
600 -O-acetyldaidzin 600 -O-malonylgenistin
1.27 0.35
daidzein
0.92
glycitein
0.03
600 -O-acetylgenistin
0.04
genistein
0.03
formononetin
0.00
total isoflavones
19.73
group was significantly reduced throughout the administration period. The bone marrow TRAP activity of the OVX-control group increased compared to that of the Sham-control group (Figure 5C). The PVEE diet significantly diminished the elevated TRAP activity in OVX mice. The serum BAP activity in the OVXcontrol group was significantly decreased compared to that in the Sham-control group. However, the PVEE diet did not alter the serum BAP activity (Figure 5D). Accordingly, these results suggest that the ovariectomy-induced incremental bone resorption was improved with a PVEEcontaining diet possibly by suppressing the osteoclast differentiation or maturation. Changes in Localization of TRAP-Positive Osteoclasts in PVEE-Fed Mice. We examined the change in osteoclast status due to ovariectomy and PVEE feeding by tissue staining. Microscopic images of osteoclasts in the femoral distal area are shown in Figure 6. TRAP-positive osteoclasts were shown to localize in the cortical bone area by costaining with TRAP and hematoxylin. OVX apparently facilitated the differentiation of preosteoclasts to TRAP-positive osteoclasts and increased the number of mature osteoclasts. The PVEE diet decreased the incremental TRAP-positive osteoclasts in OVX mice, suggesting that PVEE downregulated osteoclast differentiation. Effects of the PVEE Diet on BMD. BMD of PVEE-fed mice was examined by X-ray CT analysis. Table 2 shows time dependent change in femoral bone densities in mice fed the PVEE diet. For all parameters (cortical bone density, cancellous bone density, and total bone density), the OVX-control group had decreased bone densities compared to that of the Shamcontrol group. The PVEE diet significantly prevented the decrease in bone density in OVX mice after 4 weeks and more significantly at 8 weeks. There was no increase in bone density in the Sham mice. At 8 weeks, when the values of the Sham-control group were taken as 100%, relative cortex bone density, cancellous bone density, and total bone density of the OVX-control and the OVXPVEE group were 91% and 96%, 80% and 91%, and 90% and 95%, respectively. Accordingly, the PVEE diet effectively recovered the BMDs by 511%. 13232
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Figure 2. Chemical structures of puerarin, daidzin, and daidzein.
Figure 4. Effects of the PVEE diet on gene expression of TRAP in bone marrow cells in mice. Each data value is presented as the mean ( SD. a: p < 0.01 as compared with the Sham-control group. b: p < 0.05 as compared with the OVX-control group.
Figure 3. Effects of the PVEE diet on body weight (A) and food intake (B) in the Sham-control group (n = 6, O), Sham-PVEE group (20 mg/kg body weight/day, n = 6, b), OVX-control group (n = 6, 4), and OVXPVEE group (20 mg/kg body weight/day, n = 6, 2). Each data value is presented as the mean ( SD. a: p < 0.01 as compared with the Shamcontrol group. b: p < 0.05 as compared with the OVX-control group.
Effects of the PVEE Diet on the Spine Mass. We analyzed the effects of the PVEE diet on the backbone of mice by examining the spine using CT-3D imaging software. The CT images of the spine are shown in Figure 7. These images showed that the spinal column in OVX-control mice became more void than that in the Sham mice as indicated by less density in OVX-control mice than in Sham-control mice. The PVEE diet has improved the spine image of OVX mice to nearly 6070% compared to that of the Sham mice from the overall densitometric estimation. These results were consistent with the BMD results of the CT analysis (Table 2). Effects of the PVEE Diet on the Trabeculae of the Femur. We examined the femoral trabeculae microstructure by a stereoscopic microscope. Isolated right femurs were ground with a whetstone. The ground femur samples are shown in Figure 8. The number of trabeculae in the OVX-control group were less than that in the Sham-control group. The degree of trabecular destruction in OVX-control mice were improved in mice that consumed a PVEE diet, while there was no change in the Sham mice. Therefore, these observations indicate that the PVEE diet protects the trabecular structure from degradation by osteoclasts.
Figure 5. Effects of the PVEE diet on urinary deoxypyridinoline (A), serum TRAP activity (B), bone marrow TRAP activity (C), and serum BAP activity (D) in the Sham-control group (white), Sham-PVEE group (gray), OVX-control group (black), and OVX-PVEE group (hatched). Each data value is presented as the mean ( SD. a: p < 0.01 as compared with the Sham-control geoup. b: p < 0.05 as compared with the OVXcontrol group.
Effects of the PVEE Diet on Uterine Weight and Morphology. A uterine hypertrophy test was performed to examine
whether 8 weeks of ingestion of the PVEE diet exerted estrogenic action in vivo. The effects of ingesting PVEE diets on uterine weight and morphology are shown in Figure 9. The uterine 13233
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Figure 6. Effects of the PVEE diet on TRAP-positive osteoclasts in the Sham-control group, Sham-PVEE group, OVX-control group, and OVX-PVEE group. Representative images are shown. The images were observed at 40-fold and 400-fold magnifications. The scale bar is 500 and 100 μm, respectively.
Figure 7. Effects of the PVEE diet on the skeletal bone mass in the Sham-control group, Sham-PVEE group, OVX-control group, and OVX-PVEE group. A spine image of the mouse with a BMD closest to the mean is shown for each group.
Table 2. Effects of the PVEE Diet on BMD in Micea group
n
Cortex Bone Density 0 week 4 weeks 8 weeks
relative value at 8 weeks (%)
Sham-control
6
650 ( 19
737 ( 13
749 ( 7
100
Sham-PVEE
6
659 ( 31
734 ( 41
759 ( 43
101
OVX-control
6
618 ( 23b
681 ( 17b
688 ( 17b
91
OVX-PVEE
6
625 ( 18
703 ( 22c
727 ( 29c
96
group
n
Sham-control
6
366 ( 22
381 ( 36
399 ( 25
100
Sham-PVEE
6
372 ( 41
387 ( 42
393 ( 45
99
OVX-control
6
324 ( 24b
323 ( 19b
318 ( 16b
80
OVX-PVEE
6
332 ( 20
353 ( 32
362 ( 34c
91
group
n
Sham-control
6
568 ( 17
626 ( 30
649 ( 14
Sham-PVEE
6
573 ( 32
635 ( 43
644 ( 44
100
OVX-control
6
530 ( 22b
574 ( 13b
579 ( 16b
90
OVX-PVEE
6
538 ( 14
601 ( 27c
612 ( 33c
95
Cancellous Bone Density 0 week 4 weeks 8 weeks
c
Total Bone Density 0 week 4 weeks 8 weeks
relative value at 8 weeks (%)
Figure 8. Effects of the PVEE diet on the femur microstructure in the Sham-control group, Sham-PVEE group, OVX-control group, and OVX-PVEE group. Representative images are shown.
relative value at 8 weeks (%) 100
Each data value is presented as the mean ( SD. Relative BMD was shown when the values of Sham-control group at 8 weeks were taken 100%. b p < 0.01 as compared with the Sham-control group. c p < 0.05 as compared with the OVX-control group. a
weight was significantly decreased in OVX mice, but the PVEE diet for 8 weeks did not restore the uterine weight. No hypertrophic change in morphology was observed in the uterus. This data indicated that PVEE at this dose did not express any in vivo estrogenic action. Binding Affinity of PVEE for the ER. If isoflavones in PVEE enter into osteoclasts, some of them may exert estrogen-like activity by binding to intracellular ERs in osteoclasts. We measured the binding profile of PVEE to ER-α and -β using a commercial ELISA kit in vitro. Although the direct comparison of the affinity for ERs of crude-PVEE and pure-17β-estradiol may give some ambiguity, Figure 10 shows the binding profile of PVEE and 17β-estradiol for ER-α and -β. PVEE preferentially bound to ER-β with a little higher affinity than ER-α. However,
Figure 9. Effects of the PVEE diet on uterine weight (A). Each data value is presented as the mean ( SD. a: p < 0.01 as compared with the Sham-control group. A uterine image of the mouse that had a uterine weight closest to the mean is shown for each group (B).
PVEE bound to both ERs with affinity 5,00010,000-fold less than17β-estradiol did, suggesting that the estrogenic action of PVEE is weak, if at all. This consideration was supported by the small effect of PVEE on the uterine weight and the morphology of PVEE-fed animals as shown in Figure 7. 13234
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Figure 10. Binding affinity of PVEE with ER-α (A) and -β (B). 2 and b show the binding affinity of β-estradiol and PVEE, respectively.
’ DISCUSSION In the present study, we used ovariectomized mice as a model for postmenopausal osteoporosis. The ovariectomy operation induced an increased DPD level in the urine, increased TRAP gene expression and the enzyme activity in bone marrow, an increased number of TRAP-positive multinucleated osteoclasts in the bone marrow (Figures 46), and decreased BMD (Table 2). Oral administration of PVEE to OVX mice suppressed the elevated expression levels of TRAP mRNA in bone marrow, bone resorption markers (the urinary DPD and TRAP activity), and the number of TRAP-positive osteoclasts but did not alter serum BAP activity, a bone formation marker. These results suggested that PVEE specifically suppressed the differentiation of osteoclasts in the bone marrow. In accordance with this, the BMDs in the OVX-PVEE group after 8 weeks were significantly higher than those in the OVX-control group. Accordingly, diets containing PVEE are considered to retard bone resorption by downregulating osteoclast differentiation, thus suppressing the decrease in BMD in the OVX mice. The CT imaging data of the backbone (Figure 7) and the stereomicroscopic observation of the trabeculae (Figure 8) supported this consideration. All these results indicate that the PVEE diet protects, although not completely but partially, the bone from degradation by osteoclasts in the OVX mice. Bone vulnerabilitt depends not only on bone mass but also on bone structure. Bone intramicrostructure is an important factor for evaluating bone strength. Therefore, we visualized the femoral trabeculae. While the trabeculae of the OVX-control group decreased very much compared to that of the Shamcontrol group, the trabeculae in the OVX-PVEE group were well maintained (Figure 8). This result was consistent with the CT analysis of bone density (Table 2) and the CT images of the spine in mice (Figure 7). All these results consistently indicated that the PVEE diet improved bone strength in OVX mice. The ratio of isoflavones is generally similar between PVEE and the kudzu root, but the total isoflavone contents per unit weight are greater in the root than in the vine, although the minor components differ from each other. However, harvesting the vine is easier and economically efficient than harvesting the root.
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Then, the comparison of the effectiveness is interesting, but in Japan the kudzu root is not allowed as a food resource. Therefore, in this study kudzu vine was used as the starting material from an applicable purpose. Concerning the active species of PVEE isoflavones, puerarin was the most probable candidate since it was the most abundant (nearly 52%) of the isofuravons. The second abundant one was daizin (3.58%), followed by 600 -O-malonyldaidzin (2.52%), 600 -Oacetydaidzin (1.27%), and daidzein (0.92%). Daidzein plus modified daidzins were a total of 8.3%. The other isoflavones were below 0.5%. Genistein, which is a tyrosine kinase14with anticancer action15,16 and is reported to improve the bone mass of OVX animals,9,17 was as low as 0.03%, suggesting little contribution to the action. Thus, either puerarin or daizein plus modified daidzins was considered to be the possible candidate. Daidzein was reported to suppress bone loss in OVX mice, probably due to equol, a metabolite of daidzein according to Fujioka et al.18 Equol is known to be 100-fold more potent than daidzein in stimulating estrogenic response.19 Therefore, we tried to detect daidzein and equol by HPLC in the blood of OVX mice fed the PVEE diet. However, we detected only a small peak for daidzein and no peak for equol. Therefore, even if present, they might be below the detection limit (10 nmol/L) which is far too low for the action. Since puerarin occupies more than 50% of PVEE isoflavons, it may be probable that if puerarin is hydrolyzed in digestive organs into daidzein, the liberated daidzein, and thus equol, may participate in the PVEE action. However, it seems that this is not the case since the C-glycoside bond in puerarin is very resistant to human digestive enzymes and microflora as well as those of mice,20 although O-glycoside isoflavone, daidzin, genistin, and glycitin are not resistant.2124 In fact, we detected puerarin and puerarin-conjugates on a HPLC chromatogram of the serum of PVEE-fed mice (data not shown and will be published). This preliminary data are not fully but at least partly in agreement with the previous report that not only puerarin as the major excrement but also the small amount of daidzein, hydrodaizein, and equol were detected in urine, but not in the serum of rats.21,25 The observation by Prasain et al.26 that nonmetabolized puerarin and the small amount of its glucuronide were predominantly absorbed and detected at the early stage of feeding in the serum of puerarin-fed rats lies in the same line of our results. Furthermore, puerarin was shown to be widely distributed in tissues (kidney, liver, lung, pancreas, heart, eye, and brain) of puerarin-administrated rats.2527 On the basis of these results, they have hypothesized that the mechanism for the transport system of puerarin into major organs involved sodium-dependent glucose transporters (SGLT-1 and/or SGLT-2). Hence, most of the puerarin may presumably be absorbed to systemic circulation without deglycosylation and conjugation. However, it still remains to be fully elucidated. As the active substance in PVEE, puerarin, which constitutes more than 50% of the total isoflavones, is the most probable, though it is hard to assign PVEE function to a single substance. Puerarin(daidzein-8-C-glycoside) is a unique isoflavone only found in kudzu and differs from the other isoflavones such as as daidzin, genistin, and glycitin in that they are O-glycoside isoflavones and are converted in the gut by β-glucosidase from bacteria to their aglycons, daidzein, genistein, and glycitein, respectively.15,16,28 However, the content of isoflavones other than puerarin in PVEE are low; thus, little contribution of the corresponding aglycons is assumed. 13235
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Journal of Agricultural and Food Chemistry Concerning how PVEE diets improve the bone status of OVX mice, one of the probable explanations is that it is due to the estrogen-like action of kudzu isoflavones including puerarin, the other minor O-glycosides and the corresponding aglycons. However, this possibility could be ruled out by the results that PVEE had affinity for ER-α and -β 1/50001/10000 weaker than 17β-estradiol does (Figure 10) and that there was no hypertrophic symptoms in the uteri of PVEE-fed mice (Figure 9). These data were consistent with previous reports of the weak estrogenic activity of the kudzu root and puerarin by others.2931 Taken together, PVEE would exert little induction risk of uterus cancer by the estrogen-like action. Several previous studies examined the physiology of puerarin in vitro and in vivo. These include the protection of cells from oxidative stress,32 amelioration of learning and memory defects by long-term puerarin treatment in OVX mice,33 and improved glucose utilization by puerarin in streptozotocininduced diabetic rats.34 We hypothesized that these three beneficial actions and the present bone protection activity of PVEE (seemingly puerarin) might be brought about by the same action mechanism: one is from the phytoestrogenic action of puerarin as of daidzein and genistein, and the second is from its antioxidative nature. The low affinity of puerarin to ERs and also of daidzein and genistein and no hypertrophic action against uterine seem unfavorable to the first consideration as described above. The second consideration may be probable since intracellular oxidative stress is universally detrimental for various physiological processes related to diabetes, hypertension, and bone metabolism. In fact, we previously demonstrated that PVEE depressed TRAP gene expression of an osteoclast in RAW264.7 cells12 by downregulating NFATc1 through suppressing nuclear translocation of nuclear factor-kB, an oxidation responsive factor. More recently, Hu et al. reported that puerarin attenuated oxidative stress via the suppression of NFkB activation, an oxidation responsive factor, in LPS-induced RAW264.7 cells.35 Therefore, it may be probable that suppression of ovariectomy-induced bone loss by PVEE occurs through modification of the antioxidative NF-kB signal pathway by puerarin as one of the potential mechanisms. Consuming the PVEE diet (20 mg/kg body weight/day) for 8 weeks did not alter the body weight and morphology of the heart, lung, liver, spleen, and kidney (data not shown), indicating that this dietary dose of PVEE did not show any obvious toxicity to mice. Thus, we suppose that PVEE will be a promising functional food resource that prevents or retards osteoporosis. Now, human intervention research on the effectiveness of kudzu extracts is in progress since the safety of PVEE was confirmed by the acute and chronic toxicity evaluation and mutagenesis test in mice (will be published).
’ AUTHOR INFORMATION Corresponding Author
*Tel/Fax: +81-742-43-8067. E-mail:
[email protected]. Funding Sources
Part of this study was supported by Japan Science and Technology Agency (JST).
’ ACKNOWLEDGMENT We thank PVEE Nara Prefectural Institute of Industrial Technology.
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’ ABBREVIATIONS USED PVEE, Kudzu (Pueraria lobata) vine ethanol extracts; OVX, ovariectomized or ovariectomy; BMD, bone mineral density; HPLC, high performance liquid chromatography; RT-PCR, reverse transcriptionpolymerase chain reaction; RNA, ribonucleic acid; DNA, deoxyribonucleic acid; TRAP, tartrate-resistant acid phosphatase; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol tetraacetic acid; DTT, dithiothreitol; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; DPD, deoxypyridinoline; Cre, creatinin; BAP, bone specific alkaline phosphatase; CT, computed tomography; ER, estrogen receptor; SD, standard deviation ’ REFERENCES (1) Tucker, K. L. Osteoporosis prevention and nutrition. Curr. Osteoporosis Rep. 2009, 7, 111–117. (2) Welten, D. C.; Kemper, H. C.; Post, G. B.; van Staveren, W. A. A meta-analysis of the effect of calcium intake on bone mass in young and middle aged females and males. J. Nutr. 1995, 125, 2802– 2813. (3) Papadimitropoulos, E.; Wells, G.; Shea, B.; Gillespie, W.; Weaver, B.; Zytaruk, N.; Cranney, A.; Adachi, J.; Tugwell, P.; Josse, R.; Greenwood, C.; Guyatt, G. Meta-analyses of therapies for postmenopausal osteoporosis. VIII: Meta-analysis of the efficacy of vitamin D treatment in preventing osteoporosis in postmenopausal women. Endocr. Rev. 2002, 23, 560–569. (4) Tsugawa, N.; Shiraki, M.; Suhara, Y.; Kamao, M.; Tanaka, K.; Okano, T. Vitamin K status of healthy Japanese women: age-related vitamin K requirement for gamma-carboxylation of osteocalcin. Am. J. Clin. Nutr. 2006, 83, 380–386. (5) Koshihara, Y.; Hoshi, K.; Okawara, R.; Ishibashi, H.; Yamamoto, S. Vitamin K stimulates osteoblastogenesis and inhibits osteoclastogenesis in human bone marrow cell culture. J. Endocrinol. 2003, 176, 339–348. (6) Uesugi, T.; Toda, T.; Tsuji, K.; Ishida, H. Comparative study on reduction of bone loss and lipid metabolism abnormality in ovariectomized rats by soy isoflavones, daidzin, genistin, and glycitin. Biol. Pharm. Bull. 2001, 24, 368–372. (7) Lee, Y. B.; Lee, H. J.; Kim, K. S.; Lee, J. Y.; Nam, S. Y.; Cheon, S. H.; Sohn, H. S. Evaluation of the preventive effect of isoflavone extract on bone loss in ovariectomized rats. Biosci. Biotechnol. Biochem. 2004, 68, 1040–1045. (8) Ishida, H.; Uesugi, T.; Hirai, K.; Toda, T.; Nukaya, H.; Yokotsuka, K.; Tsuji, K. Preventive effects of the plant isoflavones, daidzin and genistin, on bone loss in ovariectomized rats fed a calcium-deficient diet. Biol. Pharm. Bull. 1998, 21, 62–66. (9) Ishimi, Y.; Arai, N.; Wang, X.; Wu, J.; Umegaki, K.; Miyaura, C.; Takeda, A.; Ikegami, S. Difference in effective dosage of genistein on bone and uterus in ovariectomized mice. Biochem. Biophys. Res. Commun. 2000, 274, 697–701. (10) Peng, N.; Prasain, J. K.; Dai, Y.; Moore, R.; Arabshahi, A.; Barnes, S.; Carlson, S.; Wyss, J. M. Chronic dietary kudzu isoflavones improve components of metabolic syndrome in stroke-prone spontaneously hypertensive rats. J. Agric. Food. Chem. 2009, 57, 7268– 7273. (11) Wang, X.; Wu, J.; Chiba, H.; Umegaki, K.; Yamada, K.; Ishimi, Y. Puerariae radix prevents bone loss in ovariectomized mice. J. Bone Miner. Metab. 2003, 21, 268–275. (12) Michihara, S.; Suzuki, S.; Tang, H.; Moriyama, T.; Kawamura, Y. Kudzu (Pueraria lobata) extracts depress bone absorption of ovariectomized mouse by downregulating NFATc1 of osteoclast. J. Clin. Biochem. Nutr. 2008, No. 43 Suppl, 141–144. (13) Dimai, H. P.; Linkhart, T. A.; Linkhart, S. G.; Donahue, L. R.; Beamer, W. G.; Rosen, C. J.; Farley, J. R.; Baylink, D. J. Alkaline phosphatase levels and osteoprogenitor cell numbers suggest bone formation may contribute to peak bone density differences between two inbred strains of mice. Bone 1998, 22, 211–216. 13236
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