Marie-Noëlle Horcajada

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.

Prevention of Bone Loss by Phloridzin, an Apple Polyphenol, in Ovariectomized Rats under Inflammation Conditions

Aging and sex hormones related changes lead to inflammatory and oxidant conditions, which are involved in the pathogenesis of osteoporosis. Recent studies have suggested that polyphenols may exert a protective effect in such conditions. We assessed the effect of phloridzin (Phlo), a flavonoid exclusively found in apple, on bone metabolism in ovariectomized (OVX) or sham-operated (SH) rats with and without inflammation. Six-month-old Wistar rats were allocated to two equal groups that received either a control diet or a diet supplemented with 0.25% Phlo for 80 days. Three weeks before necropsy, inflammation was induced by subcutaneous injection of talc in 10 animals of each group. At necropsy, ovariectomy decreased both total (T-BMD) and metaphyseal (M-BMD) femoral bone mineral density (P < 0.01). Inflammation conditions, checked by an increase in the spleen weight and α1-acid glycoprotein concentration in OVX rats, exacerbated the decrease in T-BMD (g/cm2) (as well as M-BMD) observed in castrated animals (P < 0.05). Daily Phlo intake prevented ovariectomy-induced bone loss in conditions of inflammation as shown by T-BMD and M-BMD (P < 0.05). At the diaphyseal site, BMD was improved by Phlo in OVX rats with or without inflammation (P < 0.05). These results could be explained by changes in bone remodeling as the increased urinary deoxypyridinoline excretion in OVX and OVXinf animals was prevented by the polyphenol-rich diet (P < 0.001), while plasma osteocalcin concentration was similar in all experimental groups. In conclusion, Phlo consumption may provide protection against ovariectomy-induced osteopenia under inflammation conditions by improving inflammation markers and bone resorption.

Major Phenolic Compounds in Olive Oil Modulate Bone Loss in an Ovariectomy/Inflammation Experimental Model

This study was conducted to determine whether the daily consumption for 84 days of tyrosol and hydroxytyrosol, the main olive oil phenolic compounds, and olive oil mill wastewater (OMWW), a byproduct of olive oil production, rich in micronutrients, may improve bone loss in ovariectomized rats (an experimental model of postmenopausal osteoporosis) and in ovariectomized rats with granulomatosis inflammation (a model set up for senile osteoporosis). As expected, an induced chronic inflammation provoked further bone loss at total, metaphyseal, and diaphyseal sites in ovariectomized rats. Tyrosol and hydroxytyrosol prevented this osteopenia by increasing bone formation ( p < 0.05), probably because of their antioxidant properties. The two doses of OMWW extracts had the same protective effect on bone ( p < 0.05), whereas OMWW did not reverse established osteopenia. In conclusion, polyphenol consumption seems to be an interesting way to prevent bone loss.

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.

Phenolic phytochemicals and bone

Concerning the prevention of osteoporosis, recognized as a major public health problem, nutrition may appear as an alternative strategy for optimizing health skeleton. The importance of adequate calcium and vitamin D intakes for bone health is now well documented. But, in addition to essential macro- and micronutrients, human diet contains a complex array of non-nutrient natural bioactive molecules, namely the phytochemicals that may act and protect bone. Among phytochemicals, emphasis has been so far placed upon polyphenols. Indeed, subsequent epidemiological studies have suggested associations between long-term consumption of diets rich in polyphenols and protection against chronic diseases. With respect to human health, flavonoids are the most extensively studied polyphenols. These compounds may be partly responsible for some of the positive links found between fruit and vegetables intake and higher bone mineral density in adults and children. However, no long-term intervention studies in humans have investigated the effect of specific phenolic phytochemicals on the prevention of bone loss in postmenopausal women, except for phytoestrogens (soy isoflavones, lignans). Besides, in animal models of postmenopausal osteoporosis, consumption of some dietary flavonoids has been shown to prevent ovariectomy-induced bone loss. Finally, few in vitro experiments with bone cells have reported cellular and molecular mechanisms of phytochemicals involved in bone metabolism. To date, investigations providing some evidence of a positive impact of some phytochemicals on bone metabolism are accumulating but further studies, notably clinical trials, are needed to explore the various bioactivities offered by such compounds. Anyway, it can be postulated that increased consumption of plant-derived foods, especially fruit and vegetables, may be positive in the prevention of osteoporosis.

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.

Phytonutrients for bone health during ageing.

Osteoporosis is a skeletal disease characterized by a decrease in bone mass and bone quality that predispose an individual to an increased risk of fragility fractures. Evidence demonstrating a positive link between certain dietary patterns (e.g. Mediterranean diet or high consumption of fruits and vegetables) and bone health highlights an opportunity to investigate their potential to protect against the deterioration of bone tissue during ageing. While the list of these phytonutrients is extensive, this review summarizes evidence on some which are commonly consumed and have gained increasing attention over recent years, including lycopene and various polyphenols (e.g. polyphenols from tea, grape seed, citrus fruit, olive and dried plum). Evidence to define a clear link between these phytonutrients and bone health is currently insufficient to generate precise dietary recommendations, owing to mixed findings or a scarcity in clinical data. Moreover, their consumption typically occurs within the context of a diet consisting of a mix of phytonutrients and other nutrients rather than in isolation. Future clinical trials that can apply a robust set of outcome measurements, including the determinants of bone strength, such as bone quantity (i.e. bone mineral density) and bone quality (i.e. bone turnover and bone microarchitecture), will help to provide a more comprehensive outlook on how bone responds to these various phytonutrients. Moreover, future trials that combine these phytonutrients with established bone nutrients (i.e. calcium and vitamin D) are needed to determine whether combined strategies can produce more robust effects on skeletal health.