Kudzu (Pueraria lobata) vine isoflavones, at a dose lower than the recommended daily allowance in Japan, prevents bone loss in ovariectomized mic

Abstract

As the average longevity of humans increases, the number of patients with osteoporosis has also increased. Chemoprevention for osteoporosis, especially through food intake, may extend the healthy life expectancy. We previously reported that kudzu (Pueraria lobata) vine ethanol extract (PVEE) in the diet (20 mg/kg body weight/d) suppresses bone resorption and prevents bone loss in ovariectomized mice. However, experiments with lower PVEE concentrations are necessary to determine whether PVEE is a suitable functional food resource to improve osteoporosis. This study thus examined the effects of PVEE intake (2 or 5 mg/kg body weight/day, approximately 0.07 or 0.17 mg PVEE isoflavone/d/mouse) for 6 months. The dose was calculated on the basis of the recommended daily isoflavone allowance in Japan. Both 2 and 5 mg PVEE successfully prevented ovariectomy-induced bone loss in mice. These results indicated that PVEE was effective at these doses. Thus, PVEE has the potential to be a promising resource for osteoporosis-preventing functional foods.

Introduction

Osteoporosis is characterized by bone mass reduction and micro-architectural deterioration of the bone tissue, resulting in greater bone fragility and susceptibility to fracture. Osteoporosis is mainly caused by an imbalance between osteoblastic bone formation and osteoclast resorption. This condition is especially likely to follow estrogen deficiency in menopausal women, which induces excess osteoclast activity and thus an increase in bone turnover. As the aging population has grown, bone health has become a serious problem, with the number of osteoporosis cases increasing globally. Dietary strategies to reduce the risk of postmenopausal osteoporosis are expected to extend the healthy life expectancy.

Soybean isoflavones, including daidzein and genistein, exhibit anti-osteoporotic activity in postmenopausal ovariectomized (OVX) animal models (Lee et al., 2004; Ishimi et al., 2000; Picherit et al., 2000). These isoflavones have an affinity for estrogen receptors (ERs), and specifically for ERβ (Casanova et al., 1999; Boué et al., 2003), the predominant receptor in trabecular bone but not in cortical bone (Weitzmann and Pacifici, 2006). These compounds also cause proliferation of ER-positive human breast cancer cells (MCF-7) (Boué et al., 2003; Gaete et al., 2012; Wang et al., 1996) and promote uterine hyperplasia (Ishimi et al., 2000; Rachoń et al., 2007). Hence, an excessively high isoflavone diet can potentially act through ERs to increase the risk of breast and uterine cancers. Therefore, considerable emphasis has been placed on identifying compounds with anti-osteoporotic activity, independent of ER-mediated pathways.

Kudzu (Pueraria lobata) is a creeping vine that belongs to the Leguminosae family. This plant has a global distribution, and is predominantly found in temperate climates. Puerarin is the major isoflavone in kudzu. In addition to its very weak affinity for ERα or ERβ (Michihara et al., 2012), puerarin prevents bone loss in OVX mice (Yuan et al., 2016). Furthermore, during lipopolysaccharide-induced osteolysis in osteoclast precursor RAW264.7 cells, puerarin downregulates the mRNA expression of osteoclast differentiation markers by inhibiting Akt activation (Zhang et al., 2016). Thus, kudzu-extract diets containing more puerarin than either daidzein or genistein are expected to exert anti-osteoporotic activity independent of ER-mediated pathways. Dried kudzu root (Puerariae radix) prevents bone loss in OVX mice (Wang et al., 2003). However, pharmaceutical legislation in Japan classifies kudzu root as a medicinal resource rather than a food. Therefore, we investigated whether consuming other parts of the plant would have beneficial effects in postmenopausal osteoporosis.

The isoflavone ratio is generally similar across kudzu vine ethanol extracts (PVEE) (Tanaka et al., 2011) and kudzu root extracts (Prasain et al., 2007), although the minor components differ. However, the kudzu vine has an advantage over the root as a food resource, being easier to harvest and more economical for processing into food material than the root. We previously reported that dietary PVEE (20 mg/kg body weight/d, delivering approximately 0.66 mg PVEE/d/mouse) suppresses bone resorption in OVX mice (Tanaka et al., 2011). This dosage was calculated based on the recommended isoflavone level from the Food for Specified Health Uses guidelines in Japan, with 30–40 mg supplementation/d for a 60 kg man corresponding to 0.5–0.66 mg/d. The recommended daily dietary isoflavone allowance in Japan is 15–22 mg supplementation/d for a 60 kg man corresponding to 0.25–0.37 mg/day. When we calculated the dose that exerted the same effect in mice according to body surface area, the equivalent dose was approximately 3.0–5.0 mg/kg. Therefore, further experiments are needed to examine the effectiveness of lower PVEE doses.

In this study, we investigated whether a PVEE diet (2 or 5 mg/kg body weight/day, approximately 0.07 or 0.17 mg PVEE isoflavone/d/mouse) for 6 months prevents bone loss in OVX mice.

Materials and Methods

Reagents    All chemicals used in this study were of the highest purity available.

Kudzu vine ethanol extract (PVEE)    Kudzu vines were collected from the campus of Kindai University, School of Agriculture and extracted at the Nara Prefectural Institute of Industrial Technology. Three to five volumes of ethanol was added to the kudzu vines based on their weight and then homogenized. After filtration, the extract was concentrated using an evaporator, frozen in liquid nitrogen, and then dried. The isoflavone content of PVEE has been reported previously (Tanaka et al., 2011).

Animals and diets    Slc:ddY female mice were purchased from Japan SLC (Shizuoka, Japan) and were either OVX or sham-operated at 13 weeks of age. At 1 week post-operation, sham and OVX mice were divided into six groups: Sham-0 mg PVEE (n = 6), Sham-2 mg (n = 6), Sham-5 mg (n = 6), OVX-0 mg (n = 6), OVX-2 mg (n = 6), and OVX-5 mg (n = 6). Mice in the 0 mg group were fed a control commercial diet (MF; Oriental Yeast, Tokyo, Japan) for 6 months. Mice in the 2 and 5 mg groups were fed a diet made by mixing MF powder with PVEE until reaching 2 and 5 mg isoflavone/kg body weight/d, respectively. As the amount of PVEE added was small, the base diet was not compensated with additional cornstarch. Food and water were provided ad libitum.

During the experimental period, the body weight and food intake were measured once per week. After six months, the mice were euthanized by exsanguination under anesthesia for organ collection. The uteri were harvested and weighed. Next, blood samples were collected from the heart and centrifuged to obtain the serum. All animal experiments were approved by the Kindai University Animal Care and Use Committee (approval ID: KAAG-19-003).

To examine the nutrikinetics of kudzu isoflavones, Slc:ddY female mice were purchased from Japan SLC and housed until 13 weeks of age. Mice (body weight, 38.0–44.1 g) were orally administered 400 mg/kg body weight of kudzu isoflavones suspended in 0.1% carboxymethyl cellulose sodium salt solution using a stomach tube. A vehicle solution was orally administered to the control mice, in a similar manner to the kudzu isoflavones. Blood was collected from the tail vein at 0–12 h after a single oral administration of PVEE. All samples were stored at −80 °C until analysis.

Tartrate-resistant acid phosphatase (TRAP) activity    The serum TRAP activity was measured using a colorimetric assay. Briefly, 10 µL of serum was added to 40 µL of 50 mM citric acid buffer (pH 4.6) containing 5 mM paranitrophenylphosphoric acid disodium salt and incubated for 30 min at 37 °C. The reaction was stopped with 80 µL of 0.1 N NaOH, and the absorbance at 405 nm was measured with a microplate reader (Model 680; Bio-Rad Laboratories, Hercules, CA, USA). One unit was defined as the activity that hydrolyzes 1 µmol of substrate per minute at 37 °C.

Bone-specific alkaline phosphatase (BAP) activity    Serum BAP activity was measured with the colorimetric method in the presence of the intestinal ALP inhibitor l-phenylalanine, following Dimai et al. (1998). Absorbance at 405 nm was measured with a microplate reader (Model 680).

Computed tomography (CT) analysis    The right and left femurs were scanned using an X-ray CT system (LCT-100; ALOCA, Tokyo, Japan). Tomograms were obtained at 0.1-mm intervals, and 60 slices from the end of the distal femoral area were analyzed. The cortical, cancellous, and total bone mineral densities of each femur were measured and expressed as the mean of the values from the right and left bones.

Sample preparation for the measurement of isoflavones in serum    Serum samples (10 µL) were pipetted into microtubes, followed by the addition of 10 µL of 250 µM apigenin (internal standard). Next, 40 µL of acetonitrile was added to precipitate the proteins. After vigorous mixing, all samples were centrifuged (3 000 × g for 10 min). Supernatants were diluted and filtered with syringe filters (0.45 µm) as needed. An aliquot of each sample was injected into a high-performance liquid chromatography (HPLC) system. Puerarin and daidzein levels were determined from calibration curves using authentic samples.

High-performance liquid chromatography    The HPLC system consisted of an LC-20AD pump (Shimadzu, Kyoto, Japan), a CTO-20A column oven (Shimadzu), a packed column (Cosmosil 5C₁₈-AR-II 250 × 4.6 mm, Nacalai Tesque, Kyoto, Japan), and an SPD-20A UV detector (Shimadzu). The gradient elution used was Solution A (0% acetonitrile aqueous solution, 0.1% formic acid) and Solution B (40% acetonitrile aqueous solution, 0.1% formic acid), and a gradient program of 0–50 min, 0–100% Solution B; 50–60 min, 100–100% Solution B was applied. The flow rate and column temperature were 1.0 mL/min and 37 °C, respectively. The absorbance was monitored at 260 nm.

Cell culture    The human breast adenocarcinoma cell line MCF-7 was obtained from Osaka Bioscience Institute, Japan. MCF-7 cells were incubated at 37 °C in 5% CO₂ in phenol red-free DMEM containing 10% (v/v) charcoal-stripped FBS, penicillin G, and streptomycin. This medium allowed us to examine the effects of isoflavonoids and estradiol (E2).

Cell proliferation assay    Cell proliferation was measured using the colorimetric 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Dojin, Kumamoto, Japan). Acid-isopropanol (0.04 N HCl in isopropanol) and 3% sodium lauryl sulfate were added to dissolve the reduced MTT crystals (formazan) formed in the cells. After mixing, the absorbance of each well was measured at 595 nm using a microplate reader (Model 680) with 655 nm as the reference. One day before starting this experiment, the cells were seeded in 96-well plates at a concentration of 2 × 10³ cells/well. Cells were treated with E2 as a positive control (0.1 nM) and PVEE was dissolved in dimethyl sulfoxide (DMSO). The final DMSO concentration was 0.1%. After cultivation for 4 d, MTT solution was added to the wells (50 ng/well) and the plates were incubated for 4 h, followed by colorimetric determination. The results were expressed as a percentage of the control.

Statistical analysis    Data are presented as the mean ± standard deviation (SD). Two-way analysis of variance followed by Dunnett's test was used to determine differences between the PVEE-0 and 2 mg groups of sham and OVX mice, as well as between the PVEE-0 and 5 mg groups, using the Microsoft Excel add-in software Excel Toukei 2012 (Social Survey Research Information Co. Ltd., Tokyo, Japan). Statistical significance was set at p < 0.05.

Results

Effects of long-term dietary PVEE on body weight and food intake    Changes in body weight and food intake after long-term intake of 2 or 5 mg of dietary PVEE were investigated in OVX mice to measure bone metabolism. The PVEE-2 and 5 mg diets did not affect body weight in either sham or OVX mice, and the food and PVEE intake did not change significantly in any of the mouse groups (Table 1).

Table 1. Effect of PVEE on body weight, food intake, and uterine weight.

	Sham			OVX
	0 mg	2 mg	5 mg	0 mg	2 mg	5 mg
Initial body weight (g)	33.7 ± 1.6	34.6 ± 2.6	34.2 ± 1.2	34.8 ± 2.3	35.1 ± 2.6	34.5 ± 3.4
Final body weight (g)	38.5 ± 2.1	39.2 ± 2.8	36.0 ± 1.0	43.5 ± 2.9	47.3 ± 9.0	44.1 ± 4.5
Food intake (g/d)	3.9 ± 0.1	3.8 ± 0.2	3.7 ± 0.1	3.8 ± 0.3	3.8 ± 0.2	3.7 ± 0.2
PVEE intake (g/d)	0.57 ± 0.02	0.56 ± 0.02	0.54 ± 0.02	0.56 ± 0.02	0.56 ± 0.02	0.54 ± 0.02
Uterine weight per body weight (%)	1.4 ± 0.5	1.4 ± 0.2	1.6 ± 0.4	0.2 ± 0.1	0.2 ± 0.1	0.3 ± 0.2

Effects of the PVEE diet on bone metabolic markers    We measured bone metabolic markers to determine the effects of PVEE on the bone metabolic state in mice. After measuring serum TRAP activity as a bone resorption marker, we found no significant differences between the sham groups at any PVEE concentration. However, the PVEE diet dose-dependently reduced TRAP activity in OVX mice (Fig. 1A): the OVX-5 mg group had significantly lower TRAP activity than the OVX-0 mg group (326.8 ± 37.8 vs. 233.8 ± 58.0; p < 0.05). Serum BAP activity, a bone formation marker, showed no significant changes in any of the sham and OVX mice (Fig. 1B). These results are in accordance with the conclusion of our previous study (Tanaka et al. 2011), in which PVEE suppresses bone resorption without affecting bone formation.

Fig. 1.

Effects of dietary PVEE on serum TRAP activity (A) and BAP activity (B) in the Sham-0 mg (black), Sham-2 mg (gray), Sham-5 mg (white), OVX-0 mg (black), OVX-2 mg (gray), and OVX-5 mg (white) groups. All data are presented as mean ± SD. *p < 0.05.

Effects of dietary PVEE on bone mineral density    We used X-ray CT to determine the bone mineral density in PVEE-fed mice. The OVX-2 and 5 mg groups showed significantly limited reductions in cortical bone density (508 ± 19 vs. 561 ± 24 and 508 ± 19 vs. 558 ± 29; p < 0.05), cancellous bone density (215 ± 11 vs. 245 ± 20 and 215 ± 11 vs. 254 ± 27; p < 0.05), and total bone density (422 ± 15 vs. 460 ± 24 and 422 ± 15 vs. 470 ± 31; p < 0.05) than in the OVX-0 mg group (Fig. 2). In contrast, the Sham-0 mg and Sham-2 or 5 mg groups did not differ significantly in any of the three measures of bone density among the sham mice. Therefore, low-dose PVEE improved not only bone resorption markers but also bone mineral density in OVX mice.

Fig. 2.

Effects of dietary PVEE on cortical bone density (A), cancellous bone density (B), and total bone density (C) in the Sham-0 mg (open circle), Sham-2 mg (closed triangle), Sham-5 mg (open triangle), OVX-0 mg (open circle), OVX-2 mg (closed triangle), and OVX-5 mg (open triangle) groups. All data are presented as mean ± SD. *p < 0.05.

Identification of kudzu isoflavones and their metabolites in murine serum    Thus far, our experiments demonstrated that a low-dose kudzu isoflavone diet improved bone resorption in OVX mice. To elucidate the potential mechanism, we investigated the nutrikinetics of kudzu isoflavones by examining the transition forms of orally administered kudzu isoflavones in murine blood. As can be seen from a representative HPLC chromatogram of serum at 30 min after kudzu isoflavone administration (Fig. 3), mice that ingested kudzu isoflavones exhibited some peaks that were absent in the control mice. Using liquid chromatography-mass spectrometry (data not shown), we identified two peaks: intact puerarin with an ion peak at m/z 417 [M+H]⁺ (HPLC retention time of 17.4 min) and puerarin-glucuronide with m/z 595 [M+H]⁺ (retention time of 18.6 min). Previous FAB-MS and NMR analyses concluded that 7-O-β-D-glucuronide (m/z 591) is a major metabolite in the bile of rats orally administered puerarin (Yasuda et al., 1995). The peak area that appeared to be a glucuronide conjugate of puerarin was smaller than that of puerarin. These data indicated that puerarin in PVEE translocates into the blood with little phase II reaction in murine intestinal or liver microsomes. Our HPLC results also revealed a small amount of daidzein in the serum, with a retention peak at 21.8 min. Other isoflavones detected in PVEE and their metabolites were not the major forms present in mouse blood.

Fig. 3.

Representative HPLC chromatograms of mouse serum at 30 min after administration of 400 mg/kg PVEE (upper chromatogram) and in the control (lower chromatogram). Peak retention times of 17.4, 18.6, 21.8, and 52.3 min were linked respectively to puerarin, puerarin-glucuronide, daidzein, and apigenin using authentic samples.

Additionally, HPLC analysis of isoflavones in the serum of sham and OVX mice fed PVEE for 6 months did not detect puerarin, its conjugates, or other isoflavones.

Time-dependent change of puerarin and daidzein concentrations in blood    We hypothesized that the mechanism of the anti-osteoporotic action of PVEE is strongly associated with intact puerarin, a major transformation form in mouse serum. Therefore, we measured the serum puerarin levels in mice after a single oral administration of PVEE (400 mg/kg) to determine the time of maximum puerarin concentration (T_max), maximum puerarin concentration (C_max), and its half-life (T_1/2). In addition, the amount of daidzein in the blood was quantified and compared to that of puerarin after PVEE administration. Figure 4 shows time-dependent changes in serum puerarin and daidzein levels. The T_max of serum puerarin and daidzein occurred 30 min after administration (C_max:61.9 ± 12.2 and 7.7 ± 1.0 ng/mL, respectively). Thus, the blood levels were eight times higher for puerarin than for daidzein 30 min after administration. Subsequently, serum puerarin levels decreased in a time-dependent manner and were undetectable 12 h after administration. The puerarin T_1/2 was 1.2 h.

Fig. 4.

Time-dependent variation in serum puerarin (A) and daidzein (B) concentrations after a single oral administration of PVEE (400 mg/kg) to mice. Blood was collected from the tail vein at 0, 0.5, 1, 1.5, 3, 6, and 12 h after PVEE administration. Values are mean ± SD (n = 5). N.D., below the detection limit.

Effects of PVEE on MCF-7 cell proliferation    To examine whether PVEE exerts any estrogen-like action, we explored whether it influences the proliferation of MCF-7 cells. Our results showed that four days of PVEE treatment (up to 100 µg/mL) did not enhance proliferation without cell death, but 10 nM E2 stimulated MCF-7 cell growth by 1.7 times (Fig. 5).

Fig. 5.

Effect of PVEE on MCF-7 cell proliferation. Cells were treated with PVEE and E2 for 4 d, and proliferation was measured using MTT assays. Values are presented as means ± SD for at least three replicates. *p < 0.05.

Effects of dietary PVEE on uterine weight    The uterine weight was measured to examine whether PVEE exerted estrogenic activity in vivo (Table 1). Sham mice did not exhibit hypertrophic changes in uterine weight. While an organ profile test revealed uterine atrophy in OVX mice (data not shown), 6 months of PVEE-2 and 5 mg diets did not restore uterine weight. Thus, long-term PVEE intake at the tested doses did not induce any in vivo estrogenic activity.

Discussion

This study demonstrated that 2 and 5 mg/kg PVEE intake, doses lower than the recommended daily allowance in Japan, prevented bone loss in OVX mice as effectively as the PVEE-20 mg/kg diet tested in an earlier report (Tanaka et al., 2011). The PVEE-5 mg group did not show a strong effect compared to that seen in the PVEE-20 mg group, but the effect was almost at the same level as the PVEE-20 mg group. Although serum TRAP activity was more significantly altered in the PVEE-5 mg group than in the PVEE-2 mg group, 2 mg of PVEE successfully improved bone mineral density in OVX mice. Thus, we confirmed the effectiveness of a very small dose of PVEE (2 mg/kg body weight/d) as a daily supplement for postmenopausal osteoporosis.

Although occasionally used as an ingredient in Japanese cooking, culinary experience with the kudzu vine is uncommon. Therefore, determining the safety of PVEE is important for its application in functional foods. We have already confirmed the safety of PVEE through acute and chronic toxicity evaluations and mutagenesis tests in mice (data not shown). We did not observe any abnormal behaviors or adverse effects in the mice fed PVEE diets in this study. Keyler et al. (2002) also demonstrated the safety of long-term oral administration of a Chinese herbal medicine containing puerarin as the major component. Furthermore, human intervention research has been conducted in patients with mild osteoporosis, confirming the effectiveness of kudzu extract (manuscript in preparation). Taken together, these findings indicate that PVEE is a safe functional food for preventing osteoporosis.

Puerarin was found to be a major compound in mouse serum after oral PVEE administration. Therefore, puerarin is the most likely candidate for the active component in PVEE. A previous study demonstrated that puerarin prevents bone loss in OVX mice (Yuan et al., 2016) and suppresses osteoclast differentiation in RAW264.7 pre-osteoclast cells (Yuan et al., 2016; Zhang et al., 2016). Previously, we showed that a puerarin diet (5 mg/kg body weight/d) improved incremental serum TRAP activity and mitigated the destruction of the femoral trabecular microstructure in OVX mice (Michihara et al., 2012). Therefore, although multiple active species are likely involved in the anti-osteoporotic effects of PVEE, puerarin is a major contributor.

Puerarin (daidzein-8-C-glucoside) has a different degradation profile from that of O-glycoside isoflavones. C-glycosides are not hydrolyzed (Simons et al., 2005), whereas O-glycoside isoflavones, such as daidzin, genistin, and glycitin, are easily hydrolyzed (Hur et al., 2000; Marotti et al., 2007; Simons et al., 2005). These previous reports agree with our results showing that puerarin was not hydrolyzed by β-glucosidase, β-glucuronidase, pancreatin from porcine pancreas, Caco-2 (human epithelial cell line) extracts, HepG2 (human liver cancer cell line) extracts, murine organ extracts (liver, pancreas, spleen, duodenum, jejunum, ileum, and large intestine), murine intestinal contents, or human feces (manuscript in preparation). Thus, puerarin appeared to be absorbed without being metabolized in vivo. However, other studies have contradicted this conclusion, with evidence suggesting that puerarin is degraded. For example, daidzein and equol have been detected in the urine of rats administered puerarin (Prasain et al., 2004). Additionally, human intestinal bacteria convert puerarin into daidzein and calycosin (Kim et al., 1998). Moreover, two types of bacteria that participate in the transformation of puerarin to equol have been isolated from human feces (Jin et al., 2008). Interspecific differences and variations in the assay systems may have contributed to the contradictory results of this study.

Research in a rat model has indicated that daidzein and genistein, but not their glucosides, are rapidly absorbed from the stomach (Piskula et al., 1999). In humans, ingestion of daidzein and genistein results in higher plasma isoflavone concentrations (T_max:2 h) than glucoside concentrations (T_max:4 h) (Izumi et al., 2000). Here, we showed that puerarin had a much shorter T_max than daidzein, genistein, and their glucosides. After experimenting with a rat model, Barnes et al. (2011) hypothesized that sodium-dependent glucose transporter (SGLT) is involved in puerarin transport. Therefore, the rapid absorption of puerarin from the small intestine in this study may be attributed to the action of SGLT. Moreover, T_1/2 of serum puerarin is shorter than that of daidzin or genistin, and its elimination rate is higher than that of daidzin or genistin (Izumi et al., 2000). These data may explain why puerarin could not be detected in the serum 6 months after intake. The rapid elimination rate of puerarin from animal tissues could contribute to the lack of side effects of oral administration.

The mechanism underlying the suppression of osteoclastogenesis by puerarin is of great interest. Puerarin can potentially act via nonestrogenic or antioxidative actions. E2 treatment protects against OVX-induced bone loss in animal models (Pennypacker et al., 2011; Guo et al., 2009; Modder et al., 2004). Some reports have suggested that isoflavones act as estrogen mimetics (Choi et al., 2008; Vitale et al., 2013). In OVX mice, dietary PVEE did not improve estrogen deficiency-induced uterine atrophy, whereas E2 injection reversed uterine weight loss (Kim et al., 2014; Modder et al., 2004; Chiba et al., 2003). Thus, PVEE behaves differently than E2. Moreover, PVEE treatment did not enhance proliferation without inducing cell death, whereas E2 stimulated MCF-7 cell growth. Our previous research also found that PVEE has a 1/5 000–1/10 000 weaker affinity for ER-α/β than 17β-estradiol (Tanaka et al., 2011). Two sequential processes essential for osteoclastogenesis are the activation of nuclear factor-kappa B (NF-κB), an oxidation-responsive factor, and RANK-RANKL signaling (Ono and Nakashima, 2018). Several studies have examined the protective action of puerarin against oxidative stress (Jeon et al., 2020; Wu et al., 2021; Zhou et al., 2014), including our study showing that puerarin treatment suppressed NF-κB translocation to the nucleus in TNFα+CaPO₄-induced RAW264.7 cells (Tanaka et al., 2016). Similarly, puerarin attenuated oxidative stress via suppressing NF-κB activation in RANKL-induced RAW264.7 cells (Xiao et al., 2020). Therefore, puerarin may act through an estrogen-independent pathway to downregulate NF-κB signaling. Further experiments are necessary to verify these hypotheses regarding their underlying mechanisms.

This experiment was conducted to examine the effective dose of PVEE for improving bone metabolism, which is the preliminary stage for human intervention trials. Puerarin was used as a positive control in this study. In addition, we used E2, not puerarin alone, as a positive control in cell experiments because the estrogenic and estrogen-like activity of PVEE was demonstrated. However, it is important to compare the action of PVEE and puerarin alone to identify the active compounds. Therefore, these comparative studies are considered limitations of this study.

A previous study demonstrated that daidzein prevents ovariectomy-induced bone loss in rats (Picherit et al., 2000). Because intact daidzein, although lower than the amount of puerarin, was detected in the blood of PVEE-fed mice, it is a second candidate component for the prevention of osteoporosis. However, this study did not compare the effects of daidzein and puerarin on bone metabolism, which is a limitation of this study.

PVEE is composed of approximately 80% fiber, 10% puerarin, 3.6% daidzein, 2.5% 6″-O-malonyldaidzein, and other minor isoflavones (Tanaka et al., 2011). In this study, isoflavones other than puerarin and daidzein detected in PVEE and their metabolites were not the major forms present in mouse blood. Even if present, they may be below the detection limit (10 nmol/L), which is far too low for the anti-osteoporotic action of PVEE.

In conclusion, PVEE is a promising functional food for prevention of postmenopausal osteoporosis. However, further studies are needed to clarify the molecular mechanisms underlying the anti-osteoporotic action of PVEE.

Conflict of interest    There are no conflicts of interest to declare.

Acknowledgements    This work was partially supported by a Grant-in-Aid for the Collaboration of Regional Entities for the Nara Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence (2006–2010) from the Japan Science and Technology Agency. The nutrikinetic study of kudzu isoflavones was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grants-in-Aid for Scientific Research), grant number JP20K11583 (2020–2023).

References

•  Barnes,  S.,  Prasain,  J.,  D'Alessandro,  T.,  Arabshahi,  A.,  Botting,  N.,  Lila,  M.A.,  Jackson,  G.,  Janle,  E.M., and  Weaver,  C.M. (2011). The metabolism and analysis of isoflavones and other dietary polyphenols in foods and biological systems. Food Funct., 2, 235-244.
•  Boué,  S.M.,  Wiese,  T.E.,  Nehls,  S.,  Burow,  M.E.,  Elliott,  S.,  Carter-Wientjes,  C.H.,  Shih,  B.Y.,  McLachlan,  J.A., and  Cleveland,  T.E. (2003). Evaluation of the estrogenic effects of legume extracts containing phytoestrogens. J. Agric. Food Chem., 51, 2193-2199.
•  Casanova,  M.,  You,  L.,  Gaido,  K.W.,  Archibeque-Engle,  S.,  Janszen,  D.B., and  Heck,  H.A. (1999). Developmental effects of dietary phytoestrogens in Sprague-Dawley rats and interactions of genistein and daidzein with rat estrogen receptors alpha and beta in vitro. Toxicol. Sci., 51, 236-244.
•  Chiba,  H.,  Uehara,  M.,  Wu,  J.,  Wang,  X.,  Masuyama,  R.,  Suzuki,  K.,  Kanazawa,  K., and  Ishimi,  Y. (2003) Hesperidin, a citrus flavonoid, inhibits bone loss and decreases serum and hepatic lipids in ovariectomized mice. J. Nutr., 133, 1892-1897.
•  Choi,  S.Y.,  Ha,  T.Y.,  Ahn,  J.Y.,  Kim,  S.R.,  Kang,  K.S.,  Hwang,  I.K., and  Kim,  S. (2008). Estrogenic activities of isoflavones and flavones and their structure-activity relationships. Planta Med., 74, 25-32.
•  Dimai,  HP.,  Linkhart,  T.A.,  Linkhart,  S.G.,  Donahue,  L.R.,  Beamer,  W.G.,  Rosen,  C.J.,  Farley,  J.R., and  Baylink,  D.J. (1998). Alkaline phosphatase levels and osteoprogenitor cell numbers suggest bone formation may contribute to peak bone density differences between two inbred strains of mice. Bone, 22, 211-216.
•  Gaete,  L.,  Tchernitchin,  A.N.,  Bustamante,  R.,  Villena,  J.,  Lemus,  I.,  Gidekel,  M.,  Cabrera,  G., and  Astorga,  P. (2012). Daidzein-estrogen interaction in the rat uterus and its effect on human breast cancer cell growth. J. Med. Food, 15, 1081-1090.
•  Guo,  H.Y.,  Jiang,  L.,  Ibrahim,  S.A.,  Zhang,  L.,  Zhang,  H.,  Zhang,  M., and  Ren,  F.Z. (2009). Orally administered lactoferrin preserves bone mass and microarchitecture in ovariectomized rats. J. Nutr., 139, 958-964.
•  Hur,  H.G.,  Lay,  J.O Jr.,  Beger,  R.D.,  Freeman,  J.P., and  Rafii,  F. (2000). Isolation of human intestinal bacteria metabolizing the natural isoflavone glycosides daidzin and genistin. Arch. Microbiol., 174, 422-428.
•  Ishimi,  Y.,  Arai,  N.,  Wang,  X.,  Wu,  J.,  Umegaki,  K.,  Miyaura,  C.,  Takeda,  A., and  Ikegami,  S. (2000). Difference in effective dosage of genistein on bone and uterus in ovariectomized mice. Biochem. Biophys. Res. Commun., 274, 697-701.
•  Izumi,  T.,  Piskula,  M.K,  Osawa,  S.,  Obata,  A.,  Tobe,  K.,  Saito,  M.,  Kataoka,  S.,  Kubota,  Y., and  Kikuchi,  M (2000). Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. J. Nutr. 130, 1695-1699.
•  Jeon,  Y.D.,  Lee,  J.H.,  Lee,  Y.M., and  Kim,  D.K. (2020). Puerarin inhibits inflammation and oxidative stress in dextran sulfate sodium-induced colitis mice model. Biomed. Pharmacother., 124, 109847.
•  Jin,  J.S.,  Nishihata,  T.,  Kakiuchi,  N., and  Hattori,  M. (2008). Biotransformation of C-glucosylisoflavone puerarin to estrogenic (3S)-equol in co-culture of two human intestinal bacteria. Biol. Pharm. Bull., 31, 1621-1625.
•  Keyler,  D.E.,  Baker,  J.I.,  Lee,  D.Y.,  Overstreet,  D.H.,  Boucher,  T.A., and  Lenz,  S.K. (2002). Toxicity study of an antidipsotropic Chinese herbal mixture in rats: NPI-028. J. Altern.Complem.Med., 8, 175-83.
•  Kim,  J.H.,  Meyers,  M.S.,  Khuder,  S.S.,  Abdallah,  S.L.,  Muturi,  H.T.,  Russo,  L.,  Tate,  C.R.,  Hevener,  A.L.,  Najjar,  S.M.,  Leloup,  C., and  Mauvais-Jarvis,  F. (2014). Tissue-selective estrogen complexes with bazedoxifene prevent metabolic dysfunction in female mice. Mol.Metab., 3, 177-190.
•  Kim,  D.H.,  Yu,  K.U.,  Bae,  E.A., and  Han,  M.J. (1998). Metabolism of puerarin and daidzin by human intestinal bacteria and their relation to in vitro cytotoxicity. Biol. Pharm. Bull., 21, 628- 630.
•  Lee,  Y.B.,  Lee,  H.J.,  Kim,  K.S.,  Lee,  J.Y.,  Nam,  S.Y.,  Cheon,  S.H., and  Sohn,  H.S. (2004). Evaluation of the preventive effect of isoflavone extract on bone loss in ovariectomized rats. Biosci. Biotechnol. Biochem., 68, 1040-1045.
•  Marotti,  I.,  Bonetti,  A.,  Biavati,  B.,  Catizone,  P., and  Dinelli,  G. (2007). Biotransformation of common bean (Phaseolus vulgaris L.) flavonoid glycosides by bifidobacterium species from human intestinal origin. J. Agric. Food Chem., 55, 3913-3919.
•  Michihara,  S.,  Tanaka,  T.,  Uzawa,  Y.,  Moriyama,  T., and  Kawamura,  Y. (2012). Puerarin exerted anti-osteoporotic action independent of estrogen receptor-mediated pathway. J. Nutr. Sci. Vitaminol., 58, 202-209.
•  Modder,  U.I.,  Riggs,  B.L.,  Spelsberg,  T.C.,  Fraser,  D.G.,  Atkinson,  E.J.,  Arnold,  R., and  Khosla,  S. (2004). Dose-response of estrogen on bone versus the uterus in ovariectomized mice. Eur. J. Endocrinol., 151, 503-510.
•  Ono,  T. and  Nakashima,  T. (2018). Recent advances in osteoclast biology. Histochem. Cell Biol., 149, 325-341.
•  Pennypacker,  B.L.,  Duong,  L.T.,  Cusick,  T.E.,  Masarachia,  P.J.,  Gentile,  M.A.,  Gauthier,  J.Y.,  Black,  W.C.,  Scott,  B.B.,  Samadfam,  R.,  Smith,  S.Y., and  Kimmel,  D.B. (2011). Cathepsin K inhibitors prevent bone loss in estrogen-deficient rabbits. J. Bone Miner. Res., 26, 252-262.
•  Picherit,  C.,  Coxam,  V.,  Bennetau-Pelissero,  C.,  Kati-Coulibaly,  S.,  Davicco,  M.J.,  Lebecque,  P., and  Barlet,  J.P. (2000). Daidzein is more efficient than genistein in preventing ovariectomy-induced bone loss in rats. J. Nutr., 130, 1675-1681.
•  Piskula,  M.K.,  Yamakoshi,  J., and  Iwai,  Y. (1999). Daidzein and genistein but not their glucosides are absorbed from the rat stomach. FEBS Lett., 26, 447, 287-91.
•  Prasain,  J.K.,  Reppert,  A.,  Jones,  K.,  Moore,  D.R. 2nd,  Barnes,  S., and  Lila,  M.A. (2007). Identification of isoflavone glycosides in Pueraria lobata cultures by tandem mass spectrometry. Phytochem. Anal., 18, 50-59.
•  Prasain,  J.K.,  Jones,  K.,  Brissie,  N.,  Moore,  R.,  Wyss,  J.M., and  Barnes,  S. (2004). Identification of puerarin and its metabolites in rats by liquid chromatography-tandem mass spectrometry. J. Agric. Food Chem., 52, 3708-3712.
•  Rachoń,  D.,  Vortherms,  T.,  Seidlova-Wuttke,  D., and  Wuttke,  W. (2007). Dietary daidzein and puerarin do not affect pituitary LH expression but exert uterotropic effects in ovariectomized rats. Maturitas, 57, 161-170.
•  Simons,  A.L.,  Renouf,  M.,  Hendrich,  S., and  Murphy,  P.A. (2005). Human gut microbial degradation of flavonoids: structure-function relationships. J. Agric. Food Chem., 53, 4258-4263.
•  Tanaka,  T.,  Tang,  H.,  Yu,  F.,  Michihara,  S.,  Uzawa,  Y.,  Zaima,  N.,  Moriyama,  T., and  Kawamura,  Y. (2011). Kudzu (Pueraria lobata) vine ethanol extracts improve ovariectomy-induced bone loss in female mice. J. Agric. Food Chem., 59, 13230-13237.
•  Tanaka,  T.,  Moriyama,  T.,  Kawamura,  Y., and  Yamanouchi,  D. (2016). Puerarin suppresses Macrophage activation via antioxidant mechanisms in a CaPO₄-induced mouse model of aneurysm. J. Nutr. Sci. Vitaminol., 62, 425-431.
•  Vitale,  D.C.,  Piazza,  C.,  Melilli,  B.,  Drago,  F., and  Salomone,  S. (2013). Isoflavones: estrogenic activity, biological effect and bioavailability. Eur. J. Drug Metab. Pharmacokinet., 38, 15-25.
•  Wang,  T.T.,  Sathyamoorthy,  N., and  Phang,  J.M. (1996). Molecular effects of genistein on estrogen receptor mediated pathways. Carcinogenesis, 17, 271-275.
•  Wang,  X.,  Wu,  J.,  Chiba,  H.,  Umegaki,  K.,  Yamada,  K., and  Ishimi.  Y. (2003). Puerariae radix prevents bone loss in ovariectomized mice. J. Bone Miner. Metab., 21, 268-275.
•  Weitzmann,  M.N. and  Pacifici,  R. (2006). Estrogen deficiency and bone loss: an inflammatory tale. J. Clin. Invest., 116, 1186-1194.
•  Wu,  M.,  Yi,  D.,  Zhang,  Q.,  Wu,  T.,  Yu,  K.,  Peng,  M.,  Wang,  L.,  Zhao,  D.,  Hou,  Y., and  Wu,  G. (2021). Puerarin enhances intestinal function in piglets infected with porcine epidemic diarrhea virus. Sci. Rep., 11, 6552.
•  Xiao,  L.,  Zhong,  M.,  Huang,  Y.,  Zhu,  J.,  Tang,  W.,  Li,  D.,  Shi,  J.,  Lu,  A.,  Yang,  H.,  Geng,  D.,  Li,  H., and  Wang,  Z. (2020). Puerarin alleviates osteoporosis in the ovariectomy-induced mice by suppressing osteoclastogenesis via inhibition of TRAF6/ROS-dependent MAPK/NF-kappaB signaling pathways. Aging (Albany NY), 12, 21706-21729.
•  Yasuda,  T.,  Kano,  Y.,  Saito,  K., and  Ohsawa,  K. (1995). Urinary and biliary metabolites of puerarin in rats. Biol Pharm. Bull., 18, 300-303.
•  Yuan,  S.Y.,  Sheng,  T.,  Liu,  L.Q.,  Zhang,  Y.L.,  Liu,  X.M.,  Ma,  T.,  Zheng,  H.,  Yan,  Y.  Ishimi,  Y.,  Wang,  X.X., and  Chin,  J. (2016). Puerarin prevents bone loss in ovariectomized mice and inhibits osteoclast formation in vitro. Chin. J. Nat Med., 14, 265-269.
•  Zhang,  Y.,  Yan,  M.,  Yu,  Q.F.,  Yang,  P.F.,  Zhang,  H.D.,  Sun,  Y.H.,  Zhang,  Z.F., and  Gao,  Y.F. (2016). Puerarin prevents LPS-induced osteoclast formation and bone loss via inhibition of Akt activation. Biol. Pharm Bull., 39, 2028-2035.
•  Zhou,  Y.X.,  Zhang,  H., and  Peng,  C. (2014). Puerarin: a review of pharmacological effects. Phytother. Res., 28, 961-975.