Anna Trzeciakiewicz

Hesperidin inhibits ovariectomized-induced osteopenia and shows differential effects on bone mass and strength in young and adult intact rats

The main aim of this study was to investigate the bone-sparing effect of hesperidin, one of the main flavonoid present in oranges, in two age groups of ovariectomized female rats, compared with their intact controls. Young (3 mo) and adult (6 mo) female Wistar rats were sham operated (SH) or ovariectomized (OVX) and then pair-fed for 90 days a casein-based diet supplemented or not with 0.5% hesperidin (Hp; n = 10/group). In older rats, Hp intake led to a partial inhibition of OVX-induced bone loss, whereas a complete inhibition was obtained in younger animals. At both ages, while plasma osteocalcin concentrations were unchanged, urinary excretion of deoxypyridinoline was reduced by Hp intake, suggesting that Hp was able to slow down bone resorption. Unexpectedly, in intact young rats, Hp consumption resulted in a significant increase in bone mineral density (BMD). Indeed, 6-mo-old HpSH rats had a similar BMD to 9-mo-old nontreated SH adult rats, suggesting an accelerated bone mass gain in the young rats. In contrast, in intact adult rats, Hp did not further increase BMD but did improve their bone strength. The results of this study show a protective effect of Hp on bone loss in OVX rats of both ages without uterine stimulation and accompanied by a lipid-lowering effect. The unexpected and intriguing findings obtained in intact rats showing improved BMD in young rats and improved femoral load in adult rats merit further investigation. The bone and lipid benefits of hesperidin make it an attractive dietary agent for the management of the health of postmenopausal women.

Increased bioavailability of hesperetin-7-glucoside compared with hesperidin results in more efficient prevention of bone loss in adult ovariectomised rats.

Hesperidin (Hp), a citrus flavonoid predominantly found in oranges, shows bone-sparing effects in ovariectomised (OVX) animals. In human subjects, the bioavailability of Hp can be improved by the removal of the rhamnose group to yield hesperetin-7-glucoside (H-7-glc). The aim of the present work was to test whether H-7-glc was more bioavailable and therefore more effective than Hp in the prevention of bone loss in the OVX rat. Adult 6-month-old female Wistar rats were sham operated or OVX, then pair fed for 90 d a casein-based diet supplemented or not with freeze-dried orange juice enriched with Hp or H-7-glc at two dose equivalents of the hesperetin aglycone (0.25 and 0.5 %). In the rats fed 0.5 %, a reduction in OVX-induced bone loss was observed regarding total bone mineral density (BMD):+7.0 % in OVX rats treated with Hp (HpOVX) and +6.6 % in OVX rats treated with H-7-glc (H-7-glcOVX) v. OVX controls (P < 0.05). In the rats fed 0.25 % hesperetin equivalents, the H-7-glcOVX group showed a 6.6 % improvement in total femoral BMD v. the OVX controls (P < 0.05), whereas the Hp diet had no effect at this dose. The BMD of rats fed 0.25 % H-7-glc was equal to that of those given 0.5 % Hp, but was not further increased at 0.5 % H-7-glc. Plasma hesperetin levels and relative urinary excretion were significantly enhanced in the H-7-glc v. Hp groups, and the metabolite profile showed the absence of eriodictyol metabolites and increased levels of hesperetin sulphates. Taken together, improved bioavailability of H-7-glc may explain the more efficient bone protection of this compound.

When nutrition interacts with osteoblast function: molecular mechanisms of polyphenols

Recent research has provided insights into dietary components that may optimise bone health and stimulate bone formation. Fruit and vegetable intake, as well as grains and other plant-derived food, have been linked to decreased risk of major chronic diseases including osteoporosis. This effect has been partially attributed to the polyphenols found in these foods. Thus, it has been suggested that these compounds may provide desirable bone health benefits through an action on bone cell metabolism. The present review will focus on how some polyphenols can modulate osteoblast function and reports which cellular signalling pathways are potentially implicated. However, to date, despite numerous investigations, few studies have provided clear evidence that phenolic compounds can act on osteoblasts. Polyphenols cited in the present review seem to be able to modulate the expression of transcription factors such as runt-related transcription factor-2 (Runx2) and Osterix, NF-kappaB and activator protein-1 (AP-1). It appears that polyphenols may act on cellular signalling such as mitogen-activated protein kinase (MAPK), bone morphogenetic protein (BMP), oestrogen receptor and osteoprotegerin/receptor activator of NF-kappaB ligand (OPG/RANKL) and thus may affect osteoblast functions. However, it is also important to take in account the possible interaction of these compounds on osteoclast metabolism to better understand the positive correlation reported between the consumption of fruit and vegetables and bone mass.

Differential effects of two citrus flavanones on bone quality in senescent male rats in relation to their bioavailability and metabolism.

The effect of hesperidin (Hp) and naringin (Nar), two major citrus flavanones, on the regulation of bone metabolism was examined in male senescent rats. Twenty -month -old gonad-intact male Wistar rats received a casein-based diet supplemented with or without either 0.5% hesperidin (Hp), 0.5% naringin (Nar) or a mix of both flavanones (Hp+Nar, 0.25% each). After 3 months, daily Hp intake significantly improved femoral bone integrity as reflected by improvements in total and regional bone mineral densities (BMD) (9.7%-12.3% improvements, p<0.05) and trabecular bone volume fraction (24.3% improvement, p<0.05) at the femur compared with control group. In contrast, naringin exerted site-specific effects on BMD (10.2% improvement at the distal metaphyseal area, p<0.05) and no further benefit to bone mass was observed with the mix of flavanones. Bone resorption (DPD) was significantly attenuated by Hp and Nar given alone (40.3% and 26.8% lower compared to control, p<0.05, respectively) but not by the mixture of the two. All treatments significantly reduced expression of inflammatory markers to a similar extent (IL-6, 81.0-87.9% reduction; NO, 34.7-39.5% reduction) compared to control. Bone formation did not appear to be strongly affected by any of the treatments (no effect on osteocalcin levels, modest modulation of tibial BMP-2 mRNA). However, as previously reported, plasma lipid-lowering effects were observed with Hp and Nar alone (34.1%-45.1% lower for total cholesterol and triglycerides compared to control, p<0.05) or together (46% lower for triglycerides, p<0.05). Surprisingly the plasma circulating level of naringin (8.15μM) was >5-fold higher than that of hesperidin (1.44μM) at equivalent doses (0.5%) and a linear reduction in plasma levels was observed upon co-administration (0.25% each) indicating absence of competition for their intestinal absorption sites and metabolism. The higher efficacy of Hp at a lower plasma concentration than naringin, as well as the identification of the major circulating metabolite of hesperidin (hesperetin-7-O-glucuronide) underlines the importance of flavanone bioavailability and metabolism in their biological efficacy and suggests a structure-function relationship in the mechanism of action of the active metabolites.

Molecular mechanism of hesperetin-7-O-glucuronide, the main circulating metabolite of hesperidin, involved in osteoblast differentiation.

Citrus fruit hesperidin is hydrolyzed by gut microflora into aglycone form (hesperetin) and then conjugated mainly into glucuronides. We previously demonstrated that hesperetin enhanced osteoblast differentiation. In this study, we examined the effect of hesperetin-7-O-glucuronide (Hp7G) on primary rat osteoblast proliferation and differentiation. The impact of Hp7G on specific bone signaling pathways was explored. Osteoblasts were exposed to physiological concentrations of 1 (Hp7G1) and 10 (Hp7G10) microM of conjugate. The glucuronide did not affect proliferation but enhanced differentiation by significantly increasing alkaline phosphatase (ALP) activity from day 14 of exposure. Hp7G significantly induced mRNA expression of ALP, Runx2, and Osterix after 48 h of exposure. Moreover, phosphorylation of Smad1/5/8 was enhanced by Hp7G, while ERK1/2 remained unchanged after 48 h. Hp7G decreased RANKL gene expression. These results suggest that Hp7G may regulate osteoblast differentiation through Runx2 and Osterix stimulation, and might be implicated in the regulation of osteoblast/osteoclast communication.

Hesperetin stimulates differentiation of primary rat osteoblasts involving the BMP signalling pathway.

Hesperidin found in citrus fruits has been reported to be a promising bioactive compound for maintaining an optimal bone status in ovariectomized rodent models. In this study, we examined the capacity of hesperetin (Hp) to affect the proliferation, differentiation and mineralization of rodent primary osteoblasts. Then, the impact of Hp on signalling pathways known to be implicated in bone formation was explored. We exposed osteoblasts to physiological concentrations of 1 microM Hp (Hp1) and 10 microM Hp (Hp10). Neither proliferation nor mineralization was affected by Hp at either dose during 19 days of exposure. Hp at both doses enhanced differentiation by significantly increasing alkaline phosphatase (ALP) activity from Day 14 of exposure (Day 19: Hp1: +9%, Hp10: +14.8% vs. control; P<.05). However, Hp did not induce an obvious formation of calcium nodules. The effect of Hp10 on ALP was inhibited by addition of noggin protein, suggesting a possible action of this flavanone through the bone morphogenetic protein (BMP) pathway. Indeed, Hp10 significantly induced (1.2- to 1.4-fold) mRNA expression of genes involved in this signalling pathway (i.e., BMP2, BMP4, Runx2 and Osterix) after 48 h of exposure. This was strengthened by enhanced phosphorylation of the complex Smad1/5/8. Osteocalcin mRNA level was up-regulated by Hp only at 10 microM (2.2 fold vs. control). The same dose of Hp significantly decreased osteopontin (OPN) protein level (50% vs. control) after 14 days of culture. Our findings suggest that Hp may regulate osteoblast differentiation through BMP signalling and may influence the mineralization process by modulating OPN expression.

Dietary Protein Supplementation Increases Peak Bone Mass Acquisition in Energy-Restricted Growing Rats

Peak bone mass is a major determinant of osteoporosis pathogenesis during aging. Respective influences of energy and protein supplies on skeletal growth remains unclear. We investigated the effect of a 5-mo dietary restriction on bone status in young rats randomized into six groups (n = 10 per group). Control animals were fed a diet containing a normal (13%) (C-NP) or a high-protein content (26%) (C-HP). The other groups received a 40% protein energy-restricted diet (PER-NP and PER-HP) or a 40% energy-restricted diet (ER-NP and ER-HP). High-protein intake did not modulate bone acquisition, although a metabolic acidosis was induced and calcium retention impaired. PER and ER diets were associated with a decrease in femoral bone mineral density. The compensation for protein intake in energy-restricted conditions induced a bone sparing effect. Plasma osteocalcin (OC) and urinary deoxypyridinoline (DPD) assays revealed a decreased OC/DPD ratio in restricted rats compared with C animals, which was far more reduced in PER than in ER groups. Circulating IGF-1 levels were lowered by dietary restrictions. In conclusion, both energy and protein deficiencies may contribute to impairment in peak bone mass acquisition, which may affect skeleton strength and potentially render individuals more susceptible to osteoporosis.

Hesperidin and naringin, two main flavonoids of citrus fruit, modulate bone metabolism in gonad-intact senescent male rats

Among fruit, citrus varieties are important sources of polyphenolic compounds, which could be responsible for the health-promoting effects. In the present study the effect of hesperidin (Hp) and naringin (Nar), the two major citrus flavonoids (flavanones), in regulating bone metabolism was examined in elderly male rats. Forty-eight 20-month-old gonad-intact male Wistar rats were randomized into four groups. The control group received a casein-based diet, while the other three groups were fed for 90 d the same diet but supplemented with (g/kg) 5 Hp, 5 Nar or a mix of Hp +Nar (2.5 each). Femoral mineral density and strength and bone turnover markers (urine deoxypyridinoline and osteocalcin) were measured. Plasma and serum levels of leptin, total cholesterol, TAG, insulin, glucose, insulin-like growth factor-1, IL-6, TNFa, ferric-reducing ability power, NO and circulating flavanones were also determined. During the experiment body weights for each group were stable and were unaffected by flavanone intake. However, an effect of Hp and Hp +Nar on body composition was suggested by a decrease in plasma leptin. Post-mortem examination showed that daily Hp intake improved bone mineral density compared with the control group, for all bone sites examined. While the same pattern was observed for the animals fed the mix of flavanones, only metaphyseal density was affected by Nar consumption. Nevertheless, femoral strength was not affected by flavanones. Bone resorption was slowed down by Hp and Nar whereas accretion remained unchanged. Flavanones improved lipid profiles but no changes in plasma insulin and glucose were detected. While TNFa was undetectable in serum for all groups, the consumption of flavanones appeared to prevent the increase in serum IL-6 associated with aging-related inflammation. In these conditions a slight decrease in NO production was observed for animals consuming flavanones, suggesting modulation of the oxidative status. Finally, circulating flavanones were only detected in the plasma of rats receiving Hp and/or Nar. These results suggest that both Hp and Nar affect bone metabolism in gonad-intact senescent male rats. Moreover, blood lipid profiles are clearly improved by flavanone intake. Potential anti-inflammatory and antioxidant effects of these compounds remain to be confirmed. Proceedings of the Nutrition Society (2008), 67 (OCE), E189 doi:10.1017/S0029665108008215