Pomegranate and its derivatives can improve bone health through decreased inflammation and oxidative stress in an animal model of postmenopausal osteoporosis
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- Spilmont, M., Léotoing, L., Davicco, MJ. et al. Eur J Nutr (2014) 53: 1155. doi:10.1007/s00394-013-0615-6
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Recently, nutritional and pharmaceutical benefits of pomegranate (PG) have raised a growing scientific interest. Since PG is endowed with anti-inflammatory and antioxidant activities, we hypothesized that it may have beneficial effects on osteoporosis.
We used ovariectomized (OVX) mice as a well-described model of postmenopausal osteoporosis to study the influence of PG consumption on bone health. Mice were divided into five groups as following: two control groups sham-operated and ovariectomized (OVX CT) mice fed a standard diet, versus three treated groups OVX mice given a modified diet from the AIN-93G diet, containing 5.7 % of PG lyophilized mashed totum (OVX PGt), or 9.6 % of PG fresh juice (OVX PGj) or 2.9 % of PG lyophilized mashed peel (OVX PGp).
As expected, ovariectomy was associated with a decreased femoral bone mineral density (BMD) and impaired bone micro-architecture parameters. Consumption of PGj, PGp, or PGt induced bone-sparing effects in those OVX mice, both on femoral BMD and bone micro-architecture parameters. In addition, PG (whatever the part) up-regulated osteoblast activity and decreased the expression of osteoclast markers, when compared to what was observed in OVX CT animals. Consistent with the data related to bone parameters, PG consumption elicited a lower expression of pro-inflammatory makers and of enzymes involved in ROS generation, whereas the expression of anti-inflammatory markers and anti-oxidant actors was enhanced.
These results indicate that all PG parts are effective in preventing the development of bone loss induced by ovariectomy in mice. Such an effect could be partially explained by an improved inflammatory and oxidative status.
KeywordsPomegranate Nutritional prevention Osteoporosis Animal model Inflammation Oxidative stress
Pomegranate (Punica granatum L. Punicaceae), one of the oldest known edible fruits, represents a phytochemical reservoir with a high-potential medicinal value. This fruit, grown mainly in the Mediterranean region, has been used for centuries to treat many ailments such as parasitic and microbial infections, ulcers, diarrhea, hemorrhage, and dysentery [1, 2, 3]. This is why, over the past decade, the evaluation of nutritional and pharmaceutical benefits of pomegranate (PG) has raised a great scientific interest with more than 600 publications now available on the subject.
Pomegranate composition is very complex, and each part is built up with specific components. Indeed, the fruit can be divided in two parts: (1) the edible one, called the aril, comprising 78 % juice and 22 % seeds, which constitutes 52 % of the total fruit (w/w), and (2) the peel or pericarp, which is nonedible although it has been traditionally used in folk medicine . A large array of phytochemicals has been identified in those two parts of PG, including polyphenolic compounds such as anthocyanins (cyaniding and delphinidin) in the juice and hydrolysable tannins (ellagic acid, punicalagin, gallagic acid) in the peel . Actually, the main benefit of PG has been attributed to its unique polyphenols composition . Indeed, PG polyphenols have been shown to exhibit high anti-oxidant and anti-inflammatory capacities interesting for the prevention of several age-related diseases . In this light, the health benefits of PG consumption in preventing cancers  and cardiovascular diseases  have been widely focused. However, only a few studies have targeted the eventual benefice of PG consumption toward bone health [8, 9, 10], although in the present context of longer life expectancy, the prevalence of osteoporosis has been considerably increased. As a matter of fact, with more than 50 % of women and 20 % of men affected in the US population over 50-year old, osteoporosis, the most common bone disease, represents a major economic and public health issue , and this is true worldwide. This condition is characterized by a slow decline of bone mass and impaired micro-architecture leading to increased bone susceptibility to fracture [12, 13]. Pathology establishment involves disruption of formation/resorption balance through uncoupling osteoblasts and osteoclasts activities (Gallagher and Sai 2010; Kular, Tickner et al. 2012). Besides, the literature has linked inflammation and oxidative status establishment with bone alteration, osteoclast bone resorption being promoted while osteoblast-mediated formation is inhibited [14, 15].
Along with drugs ensuring bone homeostasis strategies, recent nutritional approaches have been developed and have revealed major interests for phenolic compounds such as flavonoids, anthocyanins, and tannins derived from vegetables and drinks (green tea, wine, etc.) [16, 17, 18]. In this context, we investigated the effect of PG consumption on bone status. So far, most of the studies targeting PG health benefits have been focused on juice or purified peel polyphenol extracts: ellagic acid , punicalagin , and anthocyanins . In our hand, we investigated the in vivo (in a murine model of postmenopausal osteoporosis) potential benefits of PG, in the context of osteoporosis prevention through different nutritional approaches: (a) juice, (b) peel, and (c) whole fruit. The objective was to elucidate whether PG should be considered as one or as parts of new strategies to better contribute to bone health preservation.
Materials and methods
Chemical composition of PG products
Chemical composition of PG totum, juice, and peel (g/100 g) of dry matter
Dry matter (g/100 g)
20.787 ± 0.640
45.969 ± 1.557
24.767 ± 1.964
6.366 ± 0.476
0.678 ± 0.162
14.507 ± 0.995
92.141 ± 1.177
0.254 ± 0.030
2.926 ± 0.309
0.237 ± 0.195
22.680 ± 0.200
39.484 ± 2.390
30.003 ± 1.788
10.851 ± 1.016
0.808 ± 0.199
Totum and mashed peel were dried using a freeze drier (CHRIST, Gamma 1–20, Germany). The yield of the lyophilized powder of the PG totum was 21.2 and 25.4 % from PG peel. All samples were frozen at −20 °C, directly after preparation and until diet formulation.
All the animal procedures were approved by the institution’s animal welfare committee (Comité d’Ethique en Matière d’Expérimentation Animale Auvergne: CEMEAA) and were conducted in accordance with the European’s guidelines for the care and use of laboratory animals (2010-63UE). The animals were housed in the animal facilities of the Human Nutrition Unit at INRA Research Center (http://www1.clermont.inra.fr/unh/telechargementinternet/ienplaquette.pdf) (Agreement no. C6334514). Surgery was performed under anesthesia, and all efforts were made to minimize suffering.
Fifty-eight-week-old female C57BL/6J mice were purchased from JANVIER (St Berthevin, France). The animals were housed in a controlled environment (12:12 h light–dark cycle, at 20–22 °C, with 50–60 % relative humidity), 1 mice per plastic cage fed ad libitum and with free access to water. After an acclimatization period of 1 week, they were randomly divided into 5 groups (n = 10 per group). Two groups were surgically ovariectomized (OVX), while the other animals were sham-operated (SH), under anesthesia. The control mice (SH CT and OVX CT) were fed with a standard diet, modified from the AIN-93G powdered diet. PG groups were fed with the same standard diet, containing different amounts of PG parts. As far as the totum group is concerned (OVX PGt), 5.7 % of the PG lyophilized totum was added to the standard diet. With regards to the juice group (OVX PGj), 9.6 % of the PG fresh juice was added and 2.9 % of the PG lyophilized mashed peel was introduced into the diet for the peel group (OVX PGp). According to the human/mouse conversion principle , the experimental dose for PG juice (550 μL/mouse/day) in the present study was set equivalent to the corresponding nutritional dose of 250 mL for a 70-kg human subject. Then, the diet content of totum and peel from PG (nonedible parts) was calculated by considering the amount of fruit needed to achieve 550 μL of juice. In this light, daily quantity of PG given to the animals represented 1.4 g/mice/day of fresh totum (considering that juice = 37.5 % of the whole fruit) or 622 μg/mouse/day of fresh peel (based on the fact that peel = 42.4 % of the whole fruit). Diets were purchased from SAFE (Scientific Animal Food and Engineering, Augy, France).
Body weight was measured every 2 days during the study period. Besides, body composition was assessed at the beginning and at the end of the study, using a QMR EchoMRI-900™ system, without any anesthesia or sedation. After the 30-day treatment was completed, blood was withdrawn under anesthesia and centrifuged (3,000 rpm for 5 min at room temperature). Serum was frozen at −80 °C. Then, the mice were killed. Liver, spleen, and uterus were weighted. Femurs and tibias were harvested and stored at −80 °C prior to investigation.
Bone micro-architecture and bone mineral density analyses
After removing soft tissues, left femurs of the 14-week-old mice were placed in PBS buffer (Phosphate-Buffer Saline, Gibco) with 10 % formaldehyde at 4 °C for 1 week. Micro-architecture (secondary spongiosa) was analyzed using X-ray radiation micro-CT (SkyScan 1072). Pictures of 1,024 × 1,024 pixels were obtained at 37 kV and 215 μA. Bone morphological analysis was performed using an eXplore CT 120 scanner (GE Healthcare, Canada). Acquisitions were processed with X-ray tube settings at 100 kV and 50 mA.
Taqman low-density arrays (TLDA)
For each experimental group, four sets of extractions were performed with two tibias pooled in each. Frozen bones were ground in liquid nitrogen to obtain a fine powder. Then, total RNA was extracted from either bone powder using TRIzol reagent (Life Technology), following the manufacturer’s instructions. RNA was converted to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems). Resulting cDNA was used for TaqMan® low-density arrays (TLDAs) (Applied Biosystems 7900 HT real-time PCR system). Relative expression values were calculated using the comparative threshold cycle (2−ΔΔCT) according to Data Assist software (Applied Biosystems); 18 S, GAPDH, and actin served as housekeeping genes.
Results are expressed as means with their standard error (SEM). All the data were analyzed using XLSTAT (ExcelStat Pro software—Microsoft Office 2007) using a two-way analysis of variance (ANOVA) to test for difference among groups. If the result was found to be significant (p < 0.05), the fisher’s multiple comparison test was then used to determine specific difference between means.
Validation of the animal model
Consumption of pomegranate and its derivatives was associated with improvement of bone mineral density in OVX mice, the ultimate biomarker for bone health
Pomegranate consumption was able to counter such an osteopenic process induced by estrogen deficiency (% change in BMD values when compared to the OVX CT animals: PGt (+11.4 %), PGj (+27.1 %) (p < 0.001), and PGp (+16.2 %) (p < 0.05)).
Pomegranate was able to partially preserve bone micro-architecture in ovariectomized mice, but not its derivatives
Significant results were also observed on trabecular bone micro-architecture parameters of distal femur, as shown in Fig. 2b, c. As expected, ovariectomy significantly impaired bone micro-architecture, as shown by a decrease in trabecular number (TbN −25.0 %; p < 0.05), bone volume (BV/TV −23.6 %; p < 0.01), bone surface (BS/TV −23.9 %; p < 0.05), and connectivity (Conn Dn−19.7 %; p < 0.01) in the OVX CT, as compared with the SH CT one. Moreover, a significant increase in trabecular separation (TbSP +26.1 %; p < 0.001) and in total porosity (Po tot +3.5 %; p < 0.01) was observed as well. Consistently with BMD data, PG was endowed with bone-sparing properties as its consumption partially prevented (p < 0.01) all these micro-architectural deteriorations induced by OVX: BV/TV (+27.8 %), TbN (+26.7 %), Conn Dn (+24.6 %), BS/TV (+22.4 %), TbSP (−10.3 %), and Po tot (−3.0 %), when compared to what was measured in the OVX CT animals. With regards to PGj and PGp, only a trend toward improved biomarkers was observed, without reaching significant values.
Pomegranate was able to improve the expression profile of specific bone markers
On another hand, while ovariectomy (OVX CT) decreased the expression of osteoblast markers (0.44-fold compared to SH CT group; p < 0.001), as shown in the Fig. 3b, TLDA analysis reveals that consumption of PG as the totum or as juice was associated with improved expression of a co-receptor implied in Wnt/β-catenin, one of major osteoclast signaling pathway: LRP5 (PGt: 1.86-fold, p < 0.001; PGj: 1.60-fold, p < 0.05 PGp: 1.19-fold compared with the OVX CT group). Nevertheless, the other major osteoblast differentiation markers that have presently been investigated were not modified by any of the studied diets.
Pomegranate improved bone inflammatory and oxidative status
To further conclude on the bone protective effect of PG linked to its consumption, we investigated the impact of PGt, PGj, and PGp on major inflammation and oxidation markers known to exacerbate bone resorption. Accordingly, a transcriptomic analysis of targeted inflammatory and oxidation markers was performed on bone tissue samples using TLDA, as previously described.
On another hand, consistently, ovariectomy led to the establishment of an oxidative stress in mice bones as shown in the Fig. 4b by an increase in enzymes implied in ROS generation (nitric oxide synthase 2: NOS2 and NADPH oxidase 4: NOX4; 2.63-fold, p < 0.001; 1.37-fold p < 0.05, respectively, in the OVX CT animals compared to what was observed in the SH CT group), while expression of glutathione peroxidase: GPx and glutathione reductase: GSR (0.68-fold, p < 0.001; 0.41-fold p < 0.05, respectively, versus SH CT) was down-regulated. Interestingly, consumption of PG, whatever the part, was able to enhance those bone anti-oxidant defenses in OVX mice. Indeed, an up-regulation of those antioxidant enzymes expression [GPx (PGt: 1.72-fold, p < 0.05; PGj: 1.72-fold; PGp: 1.46-fold; p < 0.001) and GSR (PGt: 4.76-fold; PGj: 3.92-fold; PGp: 5.47-fold; p < 0.001)] was measured, while, in contrast, the expression of ROS producing enzymes was down-regulated: NOS2 (PGt: 0.24-fold; PGj: 0.23-fold; PGp: 0.27-fold; p < 0.001) and NOX4 (PGt: 0.23-fold; PGj: 0.20-fold; PGp: 0.16-fold; p < 0.001), further supporting the bone-sparing effect of PG.
The present study demonstrates for the first time that PG (as well as its derivatives (peel and juice)) consumption may contribute to bone health during aging. Actually, all PG parts were able to prevent bone loss in a well-characterized model of postmenopausal osteoporosis by modulating bone cells differentiation, as well as inflammatory and oxidative status in bone microenvironment.
First of all, to validate our experimental model, we assessed the effect of ovariecctomy on bone health. As a matter of fact, in comparison with what was observed in the SH CT group, the OVX control animals (i.e., fed the standard diet) exhibited decreased femoral BMD values and altered bone micro-architecture, as indicated by a lower number of trabeculae (TbN), decreased bone volume (BV/TV), bone surface (BS/TV), and connectivity density (ConnDn), in addition to a higher trabecular spacing (TbSp) and increased total porosity (Po tot) (Fig. 2). This is highly relevant to the human situation after menopause and consistent with the previous data [23, 24].
With regards to the dietary intervention with PG and its derivatives, the present study carried out in OVX mice showed that consumption of any of the presently studied part of PG elicited a protective effect on both bone mass and bone micro-architecture, the two parameters mainly altered in osteoporosis. Actually, our results evidenced a greater potential of PG juice and peel in preventing BMD loss, while when given as a totum (OVX PGt), PG better counteracted bone micro-architecture impairment (Fig. 2c). Accordingly, our data are consistent with those previously published by Mori-Okamoto et al.  showing that PG extracts from seed and juice exerted estrogen activities and improved bone parameters following ovariectomy. Nevertheless, in the present study and in contrast to the previous work, PG consumption (whatever the part) exhibited a bone sparing not only by modulating bone histological parameters but also through regulation of cellular processes, as well as by improving bone inflammatory and oxidative status.
Actually, PG is known for its large amount of phytochemicals and for the specific polyphenolic composition of its different parts, including anthocyanins (cyanidin, delphinidin, mainly in the juice) and hydrolyzable tannins in the peel such as ellagic acid, punicalagin, and gallagic acid . Among the great variety of those chemicals, punicalagin and ellagic acid seem to be the two important components of PG . It was raised that such a unique polyphenols composition could explain the potential of the PG to prevent various chronic diseases associated with aging, through its high anti-oxidant and anti-inflammatory capacities [3, 26, 27, 28]. In the present experiment, it appears that PG action could imply all its components: polyphenols (anthocyanins for the juice and ellagitannins for the peel), proteins, sugars, fibers, minerals, etc., although the relationship between the chemical constituents of PG and their pharmacologic effects is still not clear . Accordingly, it appears crucial to investigate, like in the present study, the benefits of the whole fruit, considering the interactions between the various compounds in the PG matrix that could be of great importance for the final result .
Nevertheless, we demonstrated here that the juice or the peel exhibited a better action on BMD than the totum. This could be due to molecules common to both parts such as ellagic acid or punicalagin.
As a matter of fact, when considering the PG totum, those molecules could be less concentrated in the whole fruit, explaining that PG totum did not significantly impact BMD, although a trend was observed. As BMD mainly depends on hydroxyapatite content , such a result would suggest a greater calcium apposition rate in the animals given PG either under the peel-enriched diet or the juice, leading to a higher BMD. This is actually consistent with the recent published work by both  showing that a PG juice extract could increase calcium bone content during gestation in mice, even though a peel extract or a mixture of both were devoid of any significant effect.
Inversely, we demonstrated that the whole fruit had a greater impact on bone micro-architecture parameters than the juice or the peel alone. This could be explained by the molecules present in PG totum such as seed compounds (punicic acid for example) that we do not find in other parts of the fruit. As micro-architecture parameters mainly result from better bone remodeling and expansion of osteoblast pool (by proliferation, recruitment, and/or increased survival) , we could suggest that PG totum improved osteoblastogenesis, leading to an improved micro-architecture.
To further investigate cellular mechanism linked to such a bone rescue, the expression of the main markers for osteoblast and osteoclast differentiation and activity was analyzed. As expected, the osteopenia process induced by ovariectomy was associated with a down-regulation of osteoblast differentiation markers and an increase in osteoclast ones. With regards to present dietary interventions, we demonstrated that each part of PG was able to counteract such a bone loss in OVX mice (Fig. 3). Moreover, as inflammation and oxidative stress play a major role in bone remodeling by modulating bone resorption [15, 31], the expression of major markers of those processes was investigated in bone tissues (Fig. 4). In this light, our data dealing with the effect of ovariectomy control are consistent with previous publications . Indeed, we confirmed that ovariectomy enhanced inflammation and oxidative stress in bones with unbalanced expression of pro- and anti-inflammatory and oxidative stress markers . In this context, PG and its derivates were able to preserve this balance and consequently to prevent from excessive bone loss.
Nevertheless, we must stay aware that this study presents some limitations. Biomechanical and histomorphometry analyses could strengthen our data. With regards to the expression analysis of major bone, oxidative stress, and inflammation markers, proteomic investigation would allow to confirm what was observed on gene expression thanks to TLDA assessment.
In conclusion, those data suggest that PG may be used as an innovative alternative agent for the prevention of osteoporosis. However, further studies are now required to determine the detailed contribution of each nutrients, composing PG in mediating the observed bone-sparing effect, and to assess if those data, obtained in an animal model for the disease, can be extrapolated to the postmenopausal women. Nevertheless, further studies are required to fully determine the contribution of each PG parts properties on bone.
Greentech (GREENTECH SA, Saint-Beauzire, France) is kindly acknowledged for providing financial support for this study. The authors are as well grateful to (1) Paul Pilet for his assistance in collecting data of bone micro-architecture from micro-CT, (2) the people from the “Animal lab: Installation Experimentale de Nutrition” who provided every day cares to mice. This study was supported by INRA, UMR 1019, UNH, Clermont-Ferrand, France.
Conflict of interest
The authors have no conflict of interest to declare.