Plum and soy aglycon extracts superior at increasing bone calcium retention in ovariectomized Sprague Dawley rats

Abstract

Plant-derived polyphenols have been shown to influence bone turnover and bone properties in the estrogen-depleted state. We used a crossover design in ovariectomized rats (n = 16 rats for each diet) to investigate the effect of supplementation of two doses each of blueberry, plum, grape, grape seed extract, and resveratrol on bone. We tested the aglycon and glucoside forms of genistein to quantify differences in efficacy on bone calcium retention. Rats were given an intravenous dose of ⁴⁵Ca to prelabel bone, and bone calcium retention was assessed by urinary excretion of ⁴⁵Ca:Ca ratio during an intervention period compared with nonintervention. Genistein aglycon increased bone calcium retention significantly (p<0.05) more than the glucoside (22% vs 13%, respectively). Plum extract (0.45% w/w total dietary polyphenols) and resveratrol (0.2% w/w total dietary polyphenols) were also effective, increasing bone calcium retention by 20% (p=0.0153) and 14% (p=0.0012), respectively. Several polyphenolic-rich diets improved bone calcium retention.

Keywords: bone calcium retention; bone resorption; genistein aglycon; genistein glucoside; plant-derived polyphenols.

Figure 1

Study design. Forty-four rats were designated into one of three study arms. Rats in arm 1 (n = 16) received blueberry and plum fruit powder and grape seed extract to compare the effect of polyphenols in the whole fruit. Rats in arm 2 (n = 16) received whole fruit polyphenols from grape to compare against the isolated resveratrol polyphenols. Rats in arm 2 were also given genistein in the aglycon form and glycosylated soy to determine the effect of conjugation. Rats in arm 3 (n = 12) were not given a dietary botanical to establish an unperturbed excretion of ⁴⁵Ca excretion.

Figure 2

Study timeline for arrival of ovariectomized (OVX) rats, dosing with ⁴⁵Ca, and urine and blood collection pattern.

Figure 3

Plot of days vs ⁴⁵Ca:Ca ratio for (A) a control rat that did not receive a botanical diet and (B) two botanical diet periods and washout periods in one rat. Each data point represents a single 24 h urine collection. Different diets are indicated by different symbols. A regression line was fit through all of the nonintervention periods, which include baseline and washout data points. During the botanical diet periods, the residual was determined by finding the difference between the predicted value from the regression line and the experimental value.

Figure 4

Effect of dietary botanicals on bone calcium retention in ovariectomized (OVX) rats (n = 16 rats per diet) was reported as mean ± 95% confidence intervals for (A) arm 1 and (B) arm 2. A value of “0” represents no change from the regression line computed from nonintervention periods. A confidence interval that excludes “0” indicates a significant change from the regression line at α = 0.05. The * indicates significant difference (p < 0.05) between high and corresponding low dose of the same dietary botanical as determined by a t test. For soy, the * indicates significant difference (p < 0.05) between genistein aglycon and glycosylated soy.

Figure 5

Effect of dietary botanicals on urinary NTx in ovariectomized (OVX) rats was reported as mean ± 95% confidence interval. A value of “1” represents no change from the regression line computed from nonintervention periods. A confidence interval that excludes “1” indicates a significant change from the regression line at α = 0.05. Contrasts were performed after ANOVA. For blueberry (n = 15 for low; n = 16 for high), plum (n = 15 for low; n = 14 for high), and grape seed extract (n = 14 for low; n = 15 for high) diets (A) * indicates a significant (p < 0.05) difference between NTx values during the low and corresponding high doses of the same diet. For soy (n = 14 for genistein aglycon; n = 15 for glycosylated soy), grape extract (n = 16 for low and high), and resveratrol (n = 16 for low and high) (B) there was a trend for NTx to be lower during the grape-high diet compared with the grape-low diet (p = 0.078). NTx, urinary collagen type 1 cross-linked N-telopeptide.

Figure 6

Effect of dietary botanicals on urinary total polyphenols in 24 h urine of ovariectomized (OVX) rats (n = 16 rats per diet). Values are reported as mean ± standard deviation with contrasts performed to detect differences between means. For blueberry, plum, and grape seed extract diets (A) * and ** indicate low is significantly different from high at p = 0.005 and p < 0.0001, respectively. All arm 1 diets were significantly different from baseline at p = 0.0015. For soy, grape, and resveratrol diets (B) * indicates that low is significantly different from high at p = 0.0002. There was a trend for total polyphenols during low to be different from high during the resveratrol-high diet with p = 0.06. All arm 2 diets were significantly different from baseline at p < 0.0001.