QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chiba, H. Articles by Ishimi, Y. Search for Related Content PubMed PubMed Citation Articles by Chiba, H. Articles by Ishimi, Y.

---

Nutrient Metabolism

Hesperidin, a Citrus Flavonoid, Inhibits Bone Loss and Decreases Serum and Hepatic Lipids in Ovariectomized Mice

Hiroshige Chiba^*, Mariko Uehara^**, Jian Wu^*,, Xinxiang Wang^*, Ritsuko Masuyama^**, Kazuharu Suzuki^**, Kazuki Kanazawa and Yoshiko Ishimi^*,,3

^* Division of Food Science and Division of Applied Food Research, National Institute of Health and Nutrition, Tokyo, Japan; ^** Department of Nutritional Science, Tokyo University of Agriculture, Tokyo, Japan; and Department of Life Science, Graduate School of Science and Technology, Kobe University, Kobe, Japan

³To whom correspondence should be addressed. E-mail: ishimi{at}nih.go.jp.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The purpose of this study was to examine whether hesperidin inhibits bone loss in ovariectomized mice (OVX), an animal model of postmenopausal osteoporosis. Forty 8-wk-old female ddY mice were assigned to five groups: a sham-operated group fed the control diet (AIN-93G), an OVX group fed the control diet, an OVX+HesA group fed the control diet containing 0.5 g/100 g hesperidin, and an OVX+HesB group fed the control diet containing 0.7 g/100 g -glucosylhesperidin and an OVX+17ß-estradiol (E₂) group fed the control diet and administered 0.03 µg E₂/d with a mini-osmotic pump. After 4 wk, the mice were killed and blood, femoral, uterine and liver were sampled immediately. Hesperidin administration did not affect the uterine weight. In OVX mice, the bone mineral density of the femur was lower than in the sham group (P < 0.05) and this bone loss was significantly prevented by dietary hesperidin or -glucosylhesperidin. The Ca, P and Zn concentrations in the femur were significantly higher in the hesperidin-fed and E₂ groups than in the OVX group. Histomorphometric analyses showed that the trabecular bone volume and trabecular thickness in the femoral distal metaphysis were markedly decreased (P < 0.05) by OVX, and -glucosylhesperidin significantly prevented this bone loss. Furthermore, hesperidin decreased the osteoclast number of the femoral metaphysis in OVX mice, as did E₂. Serum and hepatic lipids were lower in mice that consumed the hesperidin-containing diets (P < 0.05) than in the OVX group fed the control diet. These results suggest a possible role for citrus flavonoids in the prevention of lifestyle-related diseases because of their beneficial effects on bone and lipids.

KEY WORDS: • bone mineral density • estrogen • hesperidin • osteoporosis • uterus

Osteoporosis is the most common bone disease, characterized by reduced bone mineral density (BMD) and an increased risk of fracture. In particular, in postmenopausal women, osteoporosis is one of the critical disorders involving high bone turnover and bone loss attributed to estrogen deficiency (1). Although estrogen replacement therapy can prevent bone loss caused by menopause, some adverse effects such as uterine bleeding and carcinogenesis accompany it (2,3).

Several lines of evidence show that nonsteroidal, estrogen-like compounds such as phytoestrogens can prevent bone loss in osteoporotic animal models and postmenopausal women (4–8). Furthermore, certain vegetables, such as onion and Italian parsley, can prevent bone resorption in ovariectomized (OVX) rats (9,10). These vegetables are rich in flavonoids such as quercetin and rutin. In fact, rutin inhibits trabecular bone loss caused by estrogen deficiency in ovariectomized rats (9).

Among the naturally occurring citrus flavonoids, hesperidin, by pharmacological determination, is a potential anti-inflammatory agent (11). Furthermore, hesperidin may be associated with potential health benefits, such as the prevention of atherosclerosis progression, lowering cancer risks and positive effects on vaginal symptoms (12–14). Hesperidin also regulates hepatic cholesterol synthesis by inhibiting the activity of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase (15–17). Recently, Mundy et al. (18) reported that statins, cholesterol-lowering agents, induce bone formation and inhibit bone resorption both in vitro and in vivo. Furthermore, it has been confirmed that the BMD in patients treated with statins is higher than in untreated subjects (19–21). Thus, considerable attention has focused on the relationship between the inhibitory activity on HMG-CoA reductase and bone metabolism. In this study, we examined the effects of hesperidin and -glucosylhesperidin, which is 10,000 times more water soluble than hesperidin, on bone metabolism in ovariectomized mice.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animals and diets.

Eight-wk-old female ddY mice were obtained from Shizuoka Laboratory Animal Center (Shizuoka, Japan). After a 3-d adaptation period, 40 mice were either sham-operated (n = 8) or OVX (n = 32) and fed an AIN-93G control diet with corn oil instead of soybean oil (22) (Table 1). The mice were housed in individual plastic cages in a temperature- and humidity-controlled room (23 ± 1°C and 60 ± 5% relative humidity) with a 12-h light/dark cycle, and were given free access to food and distilled water. Starting on d 0, the OVX mice (n = 32) were randomly divided into four groups of 8 each. The sham group and two OVX groups were fed the control diet (AIN-93G) (Table 1). One group of OVX mice received a hesperidin-containing diet [HesA: AIN-93G diet (22) containing 5.0 g/kg hesperidin] or an -glucosylhesperidin diet (HesB: AIN-93G diet containing 7.0 g/kg -glucosylhesperidin) (Table 1) for 4 wk. Some OVX mice received subcutaneously 0.03 µg/d 17ß-estradiol (E₂) (Sigma Chemical Co., St. Louis, MO) with a mini-osmotic pump (Alza Corp., Palo Alto, CA), and fed the control diet. At the end of the experiment, the mice were killed with pentobarbital sodium (Nembutal; Dainippon Pharmaceutical Co., Osaka, Japan). In each experiment, body and uterine weights were measured, and the right and left femora were removed for the measurement of BMD and histomorphometric analyses. Both native hesperidin (hesperidin 7-rutinoside) and -glucosylhesperidin were supplied by Toyo Sugar Refining (Tokyo, Japan).

View larger version (16K): [in this window] [in a new window]   FIGURE 1 Molecular structure of -glucosylhesperidin.

Bone analysis.

The femoral bones were carefully removed at necropsy. The right femur of each mouse was used for analysis of BMD and area by dual X-ray absorptiometry (DXA, Model DCS-600R; Aloka, Tokyo, Japan). BMD was calculated by the bone mineral content of the measured area. The scanned area of the femur was equally divided into three regions (proximal, midshaft, and distal femur) to assess the regional differences.

The femora were dried overnight at 100°C, weighed and then ashed at 550°C for 48 h. The ashed samples were extracted with 1 mol/L HCl. The amounts of Ca, Mg and Zn in the femur were determined by atomic absorption spectrophotometry (Spectra AA220FS; Varian, Melbourne, Australia) (23). Phosphorus in the femur was analyzed colorimetrically (24).

The femoral cancellous bone of the distal metaphysis was analyzed two-dimensionally by use of a µCT system (µCT; Scanco Medical, Zurich, Switzerland) (25). The mean tissue volume of the scanned areas was 0.44 mm³ in the trabecular bone of the femoral distal metaphysis.

For histomorphometry of the femoral distal metaphysis, undecalcified 5-µm sections were prepared from the femora and stained with tartrate-resistant acid phosphatase (TRAP). TRAP was used as a marker for the osteoclasts. The fixed sections were incubated in an acetate buffer (0.1 mol/L sodium acetate, pH 5.0) containing naphthol AS-MX phosphate (Sigma) as a substrate and fast red violet LB salt (Sigma) as stain for the reaction product in the presence of 50 mmol/L sodium tartrate. The mean number of osteoclasts in each millimeter of trabecular bone surface was determined in the area (1.08 mm²) of the secondary spongiosa of the distal metaphysis (26). Histomorphometry was performed with a semiautomatic image system (Osteoplan II; Carl Zeiss, Thornwood, NY) linked to a light microscope (27). Histomorphometric parameters were quantified in cancellous bone tissue at the secondary spongiosa. The region in the trabecular bone within one cortical width from the endosteal surface was excluded from the measurements. Trabecular bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and the number of TRAP-positive multinucleated osteoclasts/bone surface, mm^-1 (Oc.N/BS) were measured.

Serum and liver lipids.

Lipids in the liver were extracted quantitatively with an ice-cold mixture of chloroform and methanol (2:1, v/v) by the method of Folch et al. (28). Total cholesterol (TC) and triglyceride (TG) concentrations in the liver and serum were measured by enzymatic colorimetric methods [cholesterol C test (Wako Pure Chemicals, Osaka, Japan); triglyceride E test (Wako Pure Chemicals)] (29,30). The HDL cholesterol level was measured by an enzymatic method (HDL cholesterol test; Wako Pure Chemicals) (31).

Serum hesperetin.

Serum hesperetin was measured using the method of Adlercreutz et al. (32) combined with the modified HPLC method described by Gamache et al. (33). For the recovery calculation, 20 µL of ³H-estradiol glucuronide and 10 µL of 10 mmol/L flavone (as the internal standard) were added to tubes containing 200 µL of serum and 200 µL enzyme solution. After mixing, the sample was hydrolyzed overnight at 37°C, and then unconjugated hesperetin was extracted with diethyl ether. The ether fraction was evaporated completely and dissolved in 200 µL methanol. A 10-µL sample of the solution was injected into the HPLC with electrochemical detection (Coulochem 2; ESA, Chelmsford, MA) and UV detection (SPD-10A; Shimadzu, Tokyo, Japan). Another 20 µL of the solution was used for liquid scintillation counting to determine recovery. Peaks were detected at 280 nm. The HPLC column was an MCM C₁₈ (150 x 4.6 mm I.D.; MC-Medical, Tokyo, Japan, column oven temperature, 30°C). HPLC was carried out in the mobile phase with 50 mmol/L acetate buffer (pH 4.8)/methanol/acetonitrile (50/35/15). The flow rate was 1.0 mL/min. Quantification was done by measuring the peak areas based on calibration plots of the peak area of standards at various concentrations (from 8.5 to 70 µmol/L), and corrected for losses during hydrolysis and extraction based on the recovery data.

Statistical analysis.

Data are expressed as means ± SEM. The effects of treatments were determined by use of one-way ANOVA. Differences among treatment groups were assessed by the Sheffé test (SPSS version 11.0; SPSS, Chicago, IL). Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Body and organ weights.

Initial body weights of the five groups of mice did not differ. The final body weight of the OVX group was higher than that of the sham-operated group (P < 0.05) (Table 2). Intake of hesperidin for 4 wk did not affect the body weight in OVX mice. Food intakes did not differ among the groups throughout the experiment. Uterine weight was lower (P < 0.05) in OVX mice than in sham-operated mice, indicating that they were estrogen deficient. Uterine weights of the OVX + hesperidin groups (HesA and HesB) did not differ from that of the OVX group. Uterine weight of the E₂ group was significantly higher than those of the other groups (Table 2). Liver weights among the groups did not differ.

View this table: [in this window] [in a new window]   TABLE 2 Final body weight and wet weight of the uterus in sham-operated mice (Sham) and ovariectomized (OVX) mice fed the control diet or diets containing hesperidin (HesA) or -glucosylhesperidin (HesB) or subcutaneously administered 0.03 µg/d 17ß-estradiol (E2)1

Marked bone loss occurred in the femoral cancellous bone in OVX mice, and the loss was prevented by treatment with hesperidin or -glucosylhesperidin. Administration of E₂ also prevented the bone loss in OVX mice (Fig. 2).

View larger version (47K): [in this window] [in a new window]   FIGURE 2 µCT scanning of the distal femoral metaphysis collected from sham-operated (Sham) mice; ovariectomized (OVX) mice and OVX mice fed diets containing hesperidin (HesA) or -glucosylhesperidin (HesB); and OVX mice treated with 17ß-estradiol (E2) for 4 wk. Left to right: sham-operated, OVX, OVX+HesA, OVX+HesB, OVX+E2.

View this table: [in this window] [in a new window]   TABLE 3 BMD in the femur of sham-operated mice (Sham) and ovariectomized (OVX) mice fed the control diet or diets containing hesperidin (HesA) or -glucosylhesperidin (HesB) or subcutaneously administered 0.03 µg/d 17ß-estradiol (E2)1

View this table: [in this window] [in a new window]   TABLE 4 Ca, Mg, P and Zn concentrations in the femur of sham-operated mice (Sham) and ovariectomized (OVX) mice fed the control diet or diets containing hesperidin (HesA) or -glucosylhesperidin (HesB) or subcutaneously administered 0.03 µg/d 17ß-estradiol (E2)1

Intake of HesA, HesB or E₂ restored the loss of trabecular bone in the distal femur in OVX mice fed the control diet (Fig. 3A). Histomorphometric analysis of the femoral metaphysis showed that bone volume/tissue volume (BV/TV) (Fig. 3B) and trabecular thickness (Tb.Th) in OVX mice (Fig. 3C) was much less than in sham-operated mice, whereas trabecular separation (Tb.Sp) in OVX mice (Fig. 3D) was much greater than in sham-operated mice. Intake of HesB but not HesA significantly decreased Tb.Sp in OVX mice, although HesB did not restore it to the level in the sham-operated mice (P < 0.05). Administration of E₂ prevented all of these OVX-induced changes.

View larger version (92K): [in this window] [in a new window]   FIGURE 3 Histological analysis of the trabecular bone collected from sham-operated (Sham) mice; ovariectomized (OVX) mice and OVX mice fed diets containing hesperidin (HesA) or -glucosylhesperidin (HesB); and OVX mice treated with 17ß-estradiol (E2) for 4 wk. (A) Sections of trabecular bone stained for tartrate-resistant acid phosphatase (TRAP) (x85). Left to right: sham-operated, OVX, OVX+HesA, OVX+HesB, OVX+E2. (B–E) Two-dimensional histomorphometric parameters of trabecular bone shown in A. (B) Bone volume/tissue volume (BV/TV). (C) Trabecular thickness (Tb.Th). (D) Trabecular separation (Tb.Sp). (E) Osteoclasts number/bone surface (Oc.N/BS). Values are means ± SEM, n = 8. Means without a common letter differ, P < 0.05.

Serum and liver lipids.

Serum and hepatic total cholesterol (Fig. 4A, B) and triglyceride concentrations (Fig. 4C, D) were low in the hesperidin-treated groups compared with the OVX group. The serum HDL cholesterol concentrations did not differ among the groups (Fig. 4E). The ratios of serum HDL to total cholesterol (Fig. 4F) in the groups treated with hesperidin were higher than in the OVX group (P < 0.05), and not different from that in the sham-operated group. Administration of E₂ decreased hepatic total cholesterol and triglyceride concentrations in OVX mice (Fig. 4B, D).

View larger version (52K): [in this window] [in a new window]   FIGURE 4 Serum and hepatic total cholesterol and triglyceride concentrations in sham-operated (Sham) mice; ovariectomized (OVX) mice and OVX mice fed diets containing hesperidin (HesA) or -glucosylhesperidin (HesB); and OVX mice treated with 17ß-estradiol (E2) for 4 wk. (A) Total cholesterol (TC) level in serum. (B) TC level in liver. (C) Triglycerides (TG) in serum. (D) TG in liver. (E) HDL cholesterol in serum. (F) HDL cholesterol/total cholesterol ratio in serum. Values are means ± SEM, n = 8. Means without a common letter differ, P < 0.05.

The serum concentrations of hesperidin in mice fed HesA and HesB were 5.59 ± 1.60 and 9.82 ± 2.53 µmol/L, respectively. The concentration tended to be higher in the HesB group than in the HesA group (P = 0.10). Serum hesperetin was not detected in the other three groups.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In the present study we clearly demonstrated that hesperidin not only has cholesterol-lowering effects, but also prevents bone loss in OVX mice without substantial effects on the uterus, indicating that intake of the citrus flavonoids might be useful in preventing symptoms arising from estrogen deficiency. However, because the dose of hesperidin used in this study was much higher than the usual daily intake, it is unlikely to be consumed in a normal diet.

Recently, Horcajada-Molteni et al. (10) found that rutin, which is the main flavonol in onion, inhibited OVX-induced trabecular bone loss in rats, both by slowing bone resorption and increasing osteoblastic activity. The onion extract has also been shown to inhibit bone resorption in vitro and in vivo (34,35). In this study, we demonstrated that a citrus flavonoid could prevent bone loss in OVX mice without affecting the reproductive organs. Hesperidin prevented bone loss at all three regions of the femur, indicating that this citrus flavonoid was effective on both cortical and trabecular bones (Table 3). It prevented trabecular bone resorption by a decrease in the osteoclast number at the metaphysis of the femur of OVX mice (Fig. 3B–E). Furthermore, the calcium concentration in the femur of mice fed the diet containing hesperidin was significantly higher than in OVX mice fed the control diet, and restored to a level similar to that in the sham-operated mice (Table 4). This indicates that hesperidin not only inhibits bone resorption, but also increases the mineral concentrations in the femur of OVX mice.

We assumed that the protective effect of -glucosylhesperidin on bone resorption in OVX mice would be slightly higher than that of native hesperidin but in general, the HesA and HesB groups did not differ (Fig. 3B-3E). Because the solubility of -glucosylhesperidin is 10,000 times higher than that of hesperidin, we assume that the bioavailability of the transglycosylated compound is higher than that of the corresponding aglycone. In fact, the serum concentration of hesperetin, the aglycone of hesperidin, in the mice fed the diet containing -glucosylhesperidin tended to be higher than that in the mice fed native hesperidin. These results suggest that enzymatic transglycosylation of the flavonoids with -glucosidase can increase the efficacy of the compounds by improving their water solubility.

The molecular mechanism of the inhibitory effects of hesperidin on bone resorption is not clear. Among the naturally occurring flavonoids, hesperidin has been pharmacologically evaluated as a potential anti-cancer and anti-inflammatory agent because of its antioxidant activity (11,36). Because the inhibition of osteoclastic superoxide availability reduces bone resorption (37–39), it is possible that hesperidin inhibits bone resorption by its antioxidant activity. However, the antioxidant capacity of both hesperidin and hesperetin is not as high as that of quercetin, myricetin or genistein (40). Another possibility is that hesperidin acts on bone cells through estrogen receptors (ER). ER have been found in osteoblasts and bone marrow stromal cells (41,42). The binding affinity of some flavonoids for ERß is high, although there are no data concerning the binding affinity of hesperetin for ER. Kuiper et al. (43) reported that the binding affinity of naringenin (flavanone) for ERß was 0.11, whereas those of genistein (isoflavone) and kaemferol (flavonol) were 87 and 3, respectively, when the binding affinity for E₂ was arbitrarily set at 100. The affinity of hesperetin may be low because hesperetin belongs to the same group (flavanone) as naringenin. Concerning the estrogenic activity of flavonoids evaluated by growth stimulation of estrogen-dependent human MCF-7 breast cancer cells, Breinholt and Larsen (44) hypothesized that the main feature required to confer estrogenicity was the presence of a single hydroxyl group at the 4'-position of the B-ring of the flavan nucleus. Given that hesperetin has the methoxy group at the 4'-position of the B-ring, it is not likely to show estrogenic potency through ER.

It has also been reported that a high consumption of citrus flavonoids decreases the risk of coronary heart disease, given that hesperidin lowers serum cholesterol and triglycerides in rats (15–17). Bok et al. (15) demonstrated that a mixture of naringin and hesperidin significantly lowered the levels of plasma and hepatic cholesterol and triglycerides as well as the HMG-CoA reductase activity in rats. Recently, attention has centered on the effects of cholesterol-lowering agents, statins, which are inhibitors of hepatic HMG-CoA reductase and stimulate bone formation and suppress bone resorption in animals as well as in patients with hyperlipidemia (18). It was demonstrated that statins stimulate bone formation by the production of bone morphogenic protein (18). Because hesperidin lowered serum and hepatic cholesterol and triglycerides in the present study, we hypothesize that hesperidin acts on bone by the same mechanism as that of statins. Further studies are needed to define hesperidin’s mechanism of action on bone.

In conclusion, hesperidin added to the diet not only lowered serum and hepatic cholesterol, but also inhibited bone loss by decreasing the osteoclast number in OVX mice. Examination of the effects of flavonoids on bone metabolism in osteoporotic women is required in future investigations to establish the effects of hesperidin in humans.

	ACKNOWLEDGMENTS

 
We thank Chisato Miyaura (Tokyo University of Pharmacy and Life Sciences) for assistance with the DXA analysis, and Hideki Yamato and Hisashi Murayama (Hard Tissue Research Team, Kureha Chemical Industry Co.) for their assistance with the histological analyses. We thank Toyo Sugar Refining (Tokyo) for providing hesperidin and -glucosylhesperidin.

	FOOTNOTES

 
¹ This study was supported by Special Coordination Funds for promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

² Present address: Department of Human Life and Culture, Laboratory of Biochemistry, Seitoku University, Chiba 271-8555, Japan.

⁴ BMD, bone mineral density; BV/TV, bone volume/tissue volume; E₂, 17ß-estradiol; ER, estrogen receptor; HesA, hesperidin; HesB, -glucosylhesperidin; HMG-CoA, 3-hydroxy-3-methyl-glutaryl-CoA; OC.N/BS, osteoclasts number/bone surface; OVX, ovariectomized; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; TC, total cholesterol; TRAP, tartrate-resistant acid phosphatase.

Manuscript received 3 September 2002. Initial review completed 6 October 2002. Revision accepted 21 February 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Heaney, R. P., Recker, R. R. & Saville, P. D. (1978) Menopausal changes in calcium balance performance. J. Lab. Clin. Med. 92:953-963.[Medline]

2. Genant, H. K., Baylink, D. J. & Gallagher, J. C. (1989) Estrogens in the prevention of osteoporosis in postmenopausal woman. Am. J. Obstet. Gynecol. 161:1842-1846.[Medline]

3. Recker, R. R. (1993) Current therapy for osteoporosis. J. Clin. Endocrinol. Metab. 76:14-16.[Medline]

4. Adlercreutz, H., Hamalainen, E., Gorbach, S. & Goldin, B. (1992) Dietary phytoestrogens and the menopause in Japan. Lancet 399:1233.

5. Anderson, J. W., Johnstone, B. M. & Cook-Newell, M. E. (1995) Meta-analysis of the effects of soy protein intake on serum lipids. N. Engl. J. Med. 333:276-282.[Abstract/Free Full Text]

6. Brandi, M. L. (1997) Natural and synthetic isoflavone in the prevention and treatment of chronic diseases. Calcif. Tissue Int. 61:S5-S8.

7. Ishimi, Y., Miyaura, C., Ohmura, M., Onoe, Y., Sato, T., Uchiyama, Y., Ito, M., Wang, X. X., Suda, T. & Ikegami, S. (1999) Selective effects of genistein, a soybean isoflavone, on B-lymphopoiesis and bone loss caused by estrogen deficiency. Endocrinology 140:1893-1900.[Abstract/Free Full Text]

8. Ishimi, Y., Arai, N., Wang, X. X., Wu, J., Umegaki, K., Miyaura, C., Takada, A. & Ikegami, S. (2000) Difference in effective dosage of genistein on bone and uterus in ovariectomized mice. Biochem. Biophys. Res. Commun. 274:697-701.[Medline]

9. Muhlbauer, R. C. & Li, F. (1999) Effect of vegetables on bone metabolism. Nature 401:343-344.[Medline]

10. Horcajada-Molteni, M. N., Crespy, V., Coxam, V., Davicco, M. J., Remesy, C. & Barlet, J. P. (2000) Rutin inhibits ovariectomy-induced osteopenia in rats. J. Bone Miner. Res. 15:2251-2258.[Medline]

11. Emim, J. A., Oliveira, A. B. & Lapa, A. J. (1994) Pharmacological evaluation of the anti-inflammatory activity of a citrus bioflavonoid, hesperidin, and the isoflavonoids, duartin and claussequinone, in rats and mice. J. Pharm. Pharmacol. 46:118-122.[Medline]

12. Aboobaker, V. S., Balgi, A. D. & Bhattacharya, R. K. (1994) In vivo effect of dietary factors on the molecular action of aflatoxin B1: role of non-nutrient phenolic compounds on the catalytic activity of liver fractions. In Vivo 8:1095-1098.[Medline]

13. Zhao, J., Zhang, C. Y., Xu, D. M., Huang, G. Q., Xu, Y. L., Wang, Z. Y., Fang, S. D., Chen, Y. & Gu, Y. L. (1990) The antiatherogenic effects of components isolated from pollen typhae. Thromb. Res. 57:957-966.[Medline]

14. Tanaka, T., Makita, H., Kawabata, K., Mori, H., Kakumoto, M., Satoh, K., Hara, A., Sumida, T., Tanaka, T. & Ogawa, H. (1997) Chemoprevention of azoxymethane-induced rat colon carcinogenesis by the naturally occurring flavonoids, diosmin and hesperidin. Carcinogenesis 18:957-965.[Abstract/Free Full Text]

15. Bok, S. H., Lee, S. H., Park, Y. B., Bae, K. H., Son, K. H., Jeong, T. S. & Choi, M. S. (1999) Plasma and hepatic cholesterol and hepatic activities of 3-hydroxy-3-methyl-glutaryl-CoA reductase and acyl CoA: cholesterol transferase are lower in rats fed citrus peel extract or a mixture of citrus bioflavonoids. J. Nutr. 129:1182-1185.[Abstract/Free Full Text]

16. Lee, S. H., Jeong, T. S., Park, Y. B., Kwon, Y. K., Choi, M. S. & Bok, S. H. (1999) Hypocholesterolemic effect of hesperetin mediated by inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl coenzyme A: cholesterol acyltranseferase in rats fed high-cholesterol diet. Nutr. Res. 19:1245-1258.

17. Park, Y. B., Do, K. M., Bok, S. H., Lee, M. K., Jeong, T. S. & Choi, M. S. (2001) Interactive effect of hesperidin and vitamin E supplements on cholesterol metabolism in high cholesterol-fed rats. Int. J. Vitam. Nutr. Res. 71:36-44.[Medline]

18. Mundy, G., Garrett, R., Harris, S., Chan, J., Chen, D., Rossini, G., Boyce, B., Zhao, M. & Gutierrez, G. (1999) Stimulation of bone formation in vitro and in rodents by statins. Science 286:1946-1949.[Abstract/Free Full Text]

19. Edwards, C. J., Hart, D. J. & Spector, T. D. (2000) Oral statins and increased bone-mineral density in postmenopausal women. Lancet 355:2218-2219.[Medline]

20. Chan, K. A., Andrade, S. E., Boles, M., Buist, D. S., Chase, G. A., Donahue, J. G., Goodman, M. J., Gurwitz, J. H., LaCroix, A. Z. & Platt, R. (2000) Inhibitors of hydroxymethylglutaryl-coenzyme A reductase and risk of fracture among older women. Lancet 355:185-188.[Medline]

21. Wang, P. S., Solomon, D. H., Mogun, H. & Avorn, J. (2000) HMG-CoA reductase inhibitors and the risk of hip fractures in elderly patients. J. Am. Med. Assoc. 283:3211-3216.[Abstract/Free Full Text]

22. Reeves, P. G., Nielsen, , Nielsen, F. H. & Fahey, G. C., Jr (1993) AIN-93G purified diets for laboratory rodents: final report of American Institute of Nutrition ad hoc writing committee on reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.

23. Gimblet, E. G., Marney, A. F. & Bonsnes, R. W. (1967) Determination of calcium and magnesium in serum, uterine, diet and stool by atomic absorption spectrophotometry. Clin. Chem. 13:204-214.[Abstract]

24. Gomori, G. (1942) Modification of colorimetric phosphorus determination of use with photoelectric colorimeter. J. Lab. Clin. Med. 17:955-960.

25. Ruegsegger, P., Koller, B. & Muller, R. (1996) A microtomographic system for the nondestructive evaluation of bone architecture. Calcif. Tissue Int. 58:24-29.[Medline]

26. Parfitt, A. M., Mathews, C. H., Villanueva, A. R., Kleerekoper, M., Frame, B. & Rao, D. S. (1983) Relationships between surface, volume, and thickness of iliac trabecular bone in aging and in osteoporosis. Implications for the microanatomic and cellular mechanisms of bone loss. J. Clin. Invest. 72:1396-1409.

27. Malluche, H. H., Sherman, D., Meyer, W. & Massry, S. G. (1982) A new semiautomatic method for quantitative static and dynamic bone histology. Calcif. Tissue Int. 34:439-446.[Medline]

28. Folch, J., Lees, M. & Sloane-Stanley, G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509.[Free Full Text]

29. Richmond, W. (1973) Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum. Clin. Chem. 19:1350-1356.[Abstract]

30. Spayd, R. W., Bruschi, B., Burdick, B. A., Dappen, G. M., Eikenberry, J. N., Esders, T. W., Figueras, J., Goodhue, C. T., LaRossa, D. D., Nelson, R. W., Rand, R. N. & Wu, T. W. (1978) Multilayer film elements for clinical analysis: applications to representative chemical determinations. Clin. Chem. 24:1343-1350.[Abstract/Free Full Text]

31. Ash, K. O. & Hentschel, W. M. (1978) High-density lipoproteins estimated by an enzymatic cholesterol procedure, with a centrifugal analyzer. Clin. Chem. 24:2180-2184.[Abstract/Free Full Text]

32. Adlercreutz, H., Fotsis, T., Bannwart, C., Wähälä, K., Brunow, G. & Hase, T. (1991) Isotopic dilution gas chromatographic-mass spectrometric method for the determination of lignans and isoflavonoids in human urine, including identification of genistein. Clin. Chem. Acta 199:263-278.[Medline]

33. Gamache, P. H., McCabe, D. R., Parvez, H., Parvez, A. & Acworth, I. N. (1997) The measurement of markers of oxidative damage, anti-oxidant and related compounds using HPLC and coulometric array analysis. Acworth, I. N. Naoi, M. Parvez, H. Parvez, S. eds. Progress in HPLC 1997:99-126 VSP Utrecht, The Netherlands. .

34. Ingold, P., Kneissel, M., Muhlbauer, R. C. & Gasser, J. A. (1998) Extracts from onion prevent tibial cortical and cancellous bone loss induced by a high phosphate/low protein diet in aged retired breeder rats. Bone 23(suppl.):S387.

35. Muhlbauer, R. C., Lozano, A. & Reinli, A. (2002) Onion and a mixture of vegetables, salads and herbs affect bone resorption in the rat by a mechanism independent of their base excess. J. Bone Miner. Res. 17:1230-1236.[Medline]

36. Matsubara, Y., Kumamoto, M., Iizuka, Y., Murakami, K., Okamoto, H., Miyake, H. & Yokoi, K. (1985) Structure and hypotensive effect of flavonoid glycosides in Citrus unshiu peeling. Agric. Biol. Chem. 49:909-914.

37. Key, L. L., Jr, Ries, W. L., Taylor, R. G., Hays, B. D. & Pitzer, B. L. (1990) Oxygen derived free radicals in osteoclasts: the specificity and location of the nitroblue tetrazolium reaction. Bone 11:115-119.[Medline]

38. Key, L. L., Jr, Wolf, W. C., Gundberg, C. M. & Ries, W. L. (1994) Superoxide and bone resorption. Bone 15:431-436.[Medline]

39. Manach, C., Morand, C., Crespy, V., Demigne, C., Texier, O., Regerat, F. & Remesy, C. (1998) Quercetin is recovered in human plasma as conjugated derivatives which retain antioxidant properties. FEBS Lett 426:331-336.[Medline]

40. Rice-Evans, C. A. & Miller, N. J. (1996) Antioxidant activities of flavonoids as bioactive components of food. Biochem. Soc. Trans. 24:790-795.[Medline]

41. Arts, J., Kuiper, G. G., Janssen, J. M., Gustafsson, J. A., Lowik, C. W., Pols, H. A. & Leeuwen, J. P. (1997) Differential expression of estrogen receptors and ß mRNA during differentiation of human osteoblast SV-HFO cells. Endocrinology 138:5067-5070.[Abstract/Free Full Text]

42. Air, E. L., Benoit, S. C., Clegg, D. J., Seeley, R. J. & Woods, S. C. (2002) Insulin and leptin combine additively to reduce food intake and body weight in rats. Endocrinology 143:2349-2356.[Abstract/Free Full Text]

43. Kuiper, G. G., Lemmen, J. G., Carlsson, B., Corton, J. C., Safe, S. H., van der Saag, P. T., van der Burg, B. & Gustafsson, J. A. (1998) Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor ß. Endocrinology 135:4252-4263.

44. Breinholt, V. & Larsen, J. C. (1998) Detection of weak estrogenic flavonoids using a recombinant yeast strain and a modified MCF7 cell proliferation assay. Chem. Res. Toxicol. 11:622-629.[Medline]

This article has been cited by other articles:

				B. Morin, L. A. Nichols, K. M. Zalasky, J. W. Davis, J. A. Manthey, and L. J. Holland The Citrus Flavonoids Hesperetin and Nobiletin Differentially Regulate Low Density Lipoprotein Receptor Gene Transcription in HepG2 Liver Cells J. Nutr., July 1, 2008; 138(7): 1274 - 1281. [Abstract] [Full Text] [PDF]

				M. N. Horcajada, V. Habauzit, A. Trzeciakiewicz, C. Morand, A. Gil-Izquierdo, J. Mardon, P. Lebecque, M. J. Davicco, W. S. S. Chee, V. Coxam, et al. Hesperidin inhibits ovariectomized-induced osteopenia and shows differential effects on bone mass and strength in young and adult intact rats J Appl Physiol, March 1, 2008; 104(3): 648 - 654. [Abstract] [Full Text] [PDF]

				M. R. Peluso Flavonoids Attenuate Cardiovascular Disease, Inhibit Phosphodiesterase, and Modulate Lipid Homeostasis in Adipose Tissue and Liver Experimental Biology and Medicine, September 1, 2006; 231(8): 1287 - 1299. [Abstract] [Full Text] [PDF]

				I. L. F. Nielsen, W. S. S. Chee, L. Poulsen, E. Offord-Cavin, S. E. Rasmussen, H. Frederiksen, M. Enslen, D. Barron, M.-N. Horcajada, and G. Williamson Bioavailability Is Improved by Enzymatic Modification of the Citrus Flavonoid Hesperidin in Humans: A Randomized, Double-Blind, Crossover Trial J. Nutr., February 1, 2006; 136(2): 404 - 408. [Abstract] [Full Text] [PDF]

				R. C. Muhlbauer, A. Lozano, A. Reinli, and H. Wetli Various Selected Vegetables, Fruits, Mushrooms and Red Wine Residue Inhibit Bone Resorption in Rats J. Nutr., November 1, 2003; 133(11): 3592 - 3597. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chiba, H. Articles by Ishimi, Y. Search for Related Content PubMed PubMed Citation Articles by Chiba, H. Articles by Ishimi, Y.