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Evidences of osteoporosis improvement in Wistar rats treated with Ginkgo biloba extract: A histomorphometric study of mandible and femur
Fitoterapia 81 (2010) 982–987
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Evidences of osteoporosis improvement in Wistar rats treated with Ginkgo biloba extract: A histomorphometric study of mandible and femur L.M.F. Lucinda a,⁎, B.J. Vieira a, T.T. Oliveira b, R.C.S. Sá a, V.M. Peters a, J.E.P. Reis a, M.O. Guerra a a b
Centro de Biologia da Reprodução, Federal University of Juiz de Fora, Caixa Postal 328, Zip Code 36001-970. Juiz de Fora, MG, Brazil Departamento de Bioquímica e Biologia Molecular, Federal University of Viçosa, Vila Gianetti, casa 26, Zip Code 36570-000. Viçosa, MG, Brazil
a r t i c l e
i n f o
Article history: Received 23 February 2010 Accepted in revised form 8 June 2010 Available online 25 June 2010 Keywords: Ginkgo biloba Osteoporosis Histomorphometry Sodium alendronate Rats
a b s t r a c t This study was aimed at investigating the anti-osteoporotic effects of the extract of Ginkgo biloba (EGb) in glucocorticoid-induced-osteoporosis. A significant reduction was observed in the percentage of the bone of the osteoporosis group in both the mandible and femur. The EGb group treated with 28 and 56 mg/Kg showed a significant increase in the percentage of trabecular bone (PTB) of the femur. The percentage of the alveolar bone of the mandible (PAB) had a significant increase with all doses of EGb. The treatment with EGb significantly reversed the loss of the PAB of the mandible and of the PTB of the femur. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Osteoporosis is a metabolic bone disease characterized by low bone mass and a microarchitectural deterioration of bone tissue leading to bone fragility and an increase in bone fracture [1]. Low bone mass results from genetic, nutritional and lifestyle factors, decreased estrogen levels and the use of drugs [2], such as glucocorticoids that are widely used in the treatment of chronic diseases because of their immunosuppressive and anti-inflammatory properties. The long-term use of glucocorticoids is associated with numerous side effects, such as the development of osteoporosis. This medication interacts with bone metabolism at many levels, but their principal action is to reduce osteoblast number and bone matrix synthesis. The change in osteoblast activity is followed by a fall in bone mass that is maximal over the first few moments of therapy [3]. Biphosphonates are a group of well documented drugs used in the treatment of osteoporosis, although side effects
⁎ Corresponding author. Rua Doutor Pedro de Aquino Ramos 106/503, Granbery. Zip Code: 36010-440, Juiz de Fora, MG, Brazil. Tel.: + 55 32 21023251, + 55 32 32180734; fax: + 55 32 21023251. E-mail address: [email protected] (L.M.F. Lucinda). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.06.014
like gastrointestinal intolerance [4] and osteonecrosis of the jaws [5] have been reported in some patients. The search for new therapeutic alternatives to treat osteoporosis has been under scrutiny over the years. For instance, studies with phytoestrogens (plant-derived compounds possessing estrogen-like activity) showed that they are promising alternatives to treat osteoporosis [6]. Ginkgo biloba L. (Family Ginkgoaceae) is a native plant from China, Japan and Korea where its fruits and leaves have long been used as a food source and a folk medicine. Phytochemical studies have shown a standard composition of substances in this plant comprising: 24% of phytoestrogens (kaempferol, kercetin and ishorhamnetin), 6% of terpenoids (ginkgolides and bilobilides) and less than 5 ppm of ginkgolic acid [7,8]. The main pharmacological effects of the extract of G. biloba (EGb) are vascular and tissue protection, and improvement in cognitive function [9,10] while the phytoestrogen compounds, such as quercetin, kaempferol and ishorhamnetin, were effective in inhibiting osteoclast activity in vitro [10,11]. A recent study showed that EGb stimulated osteoblast differentiation and antiosteoclastic activity in vitro [12]. These effects together with the antioxidant activity of EGb could be important in the treatment of osteoporosis, since
L.M.F. Lucinda et al. / Fitoterapia 81 (2010) 982–987
this activity protected osteoblasts from cellular damages and death when they were exposed to the action of free radicals in vitro [13]. Our previous study showed by digital radiography that EGb was effective in recovering the periodontal bone support (PBS) and in increasing the cortical thickness of the mandible of rats with glucocorticoid-induced-osteoporosis [14]. Considering the pharmacological effects of G. biloba on bone tissue and cells, and our previous results with digital radiographics [14], the present study was designed to evaluated by histomorphometric technique the effect of the EGb on mandibular and femoral bone of Wistar rats with glicocorticoid-induced-osteoporosis.
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six groups (n = 6). The control group was not submitted to osteoporosis induction. 1 osteoporosis group (dexamethasone); 2 positive control group (sodium alendronate 0.2 mg/animal/day administered intragastrically, once a day, during 30 days, after the osteoporosis induction) [15]; 3 EGb1 group (G. biloba 14 mg/kg/day); 4 EGb2 group (G. biloba 28 mg/kg/day); 5 EGb3 group (G. biloba 56 mg/kg/day); and 6 control group. The doses of EGb were administered intragastrically, once a day, during 30 days, after the osteoporosis induction. The choice of EGb doses was based on previous studies [16], which suggested an estrogenic effect (intra-uterine growth retardation) in pregnant rats with the dose of 14 mg of EGb/kg/day. The subsequent doses are multiples of the former one.
2. Methods The methodology of this work was approved by the Ethical Committee on Animal Experimentation (protocol number 58/2006, CEEA, Federal University of Juiz de Fora, MG, Brazil), which follows the international principles in ethics for animal experimentation. 2.1. Plant material EGb was supplied by JR Pharma (China-lot no. 06112216c). The quality test made by Galena Laboratory showed that the extract was composed of: 28.2% of ginkgoflavonglicosides; 8.3% of terpenolactones; 15% of quercetin glycosides; 10.9% of kaempferol glycosides; 2.3% of ishorhamnetin glycosides and less than 5 ppm of ginkgolic acids. 2.2. Sodium alendronate The solution of sodium alendronate was supplied by JR Pharma (Solution-lot no SA/ 20060501). 2.3. Animals Female Wistar rats (Rattus norvegicus Berkenhout, 1769) (50 days old and weighing approximately 100–150 g) were obtained from the vivarium of the Federal University of Juiz de Fora (UFJF), where they were born and bred. Groups of three animals were housed in clear plastic cages with stainless steel wire lids and pinewood shavings as bedding and kept in room with controlled environmental conditions (12-h light/12-h dark cycle, temperature 22 °C), on closed ventilated shelves. The animals were fed on rat chow pellets (an average of 25 g daily) and received water ad libitum. 2.4. Osteoporosis induction The osteoporosis induction was done through the intramuscular administration of dexamethasone disodium phosphate (Decadron®-4 mg/ml) at the dose level of 7 mg/kg of body weight, once a week, during five weeks, in all groups, except in the control group [15]. 2.5. Bioassay In this experiment, after the end of osteoporosis induction, 36 animals were selected at random and divided evenly into
2.6. Histomorphometry The samples of the mandible and femur were analyzed in an optical microscope system Zeiss (Hallbergmoos, Germany) for digital capturing photomicrographs. All slides were analyzed in the microscope at 250X and 400× magnifications by a pathologist. Histomorphometric analysis was conducted by only one evaluator, who was unaware of the experimental groups.
2.6.1. Histomorphometric analysis of the femur The trabecular bone tissue of the proximal epiphysis of the femur was photographed at 25× magnification. Three areas were identified: two lateral areas (A and C) and a medial one (B) as shown in Fig. 1. Each region (A, B and C) presented a total area of 16,000 μ2. Through the software Axiovision® (version 4.5 for Windows semi-automatic), the trabecular bone tissue was marked and measured with the assistance of a digital cursor pen. The delineated bone tissue area was converted into proportion of bone tissue according to the following formula: % of bone tissue = area measured ×100/16,000 μ2. After the calculation of the proportion of bone tissue from the regions A, B and C, the mean of the proportions of the femoral epiphysis was established for further statistical processing.
2.6.2. Histomorphometric analysis of the mandible The histomorphometric analysis of the alveolar bone of the mandible was performed in the interradicular septum of the second molar (Fig. 2). The measuring procedure was the same as that used in the femur analysis, with the exception that only the bone tissue limited to the interradicular septum was measured.
2.7. Statistical analysis The data were expressed by mean ± standard error mean (S.E.M.) and were analyzed for statistical significance using one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test. The control group was compared with the osteoporosis group using the Student's t-test. P b 0.05 was considered significant.
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Fig. 1. (A) Schematic drawing of the areas selected for the morphometric analysis of the trabecular bone of the femur (B) and of the interradicular alveolar bone of the mandible.
3. Results Fig. 2 shows the histological sections of the interradicular septum of the second molar in which the reduction of the mandible bone area is identified by a significant reduction of the alveolar bone. The significant reduction of the epiphiseal area of the femur is observed in Fig. 3 and identified by the lower thickness of the trabeculae bone. Both the mandible and femur presented a larger area of bone marrow in the animals with osteoporosis when they were compared to the control group. The treated groups show an increase in the alveolar bone area and reduction of the bone marrow area in the interradicular septum of the second molar. Similarly, the femur shows increase in the trabecular bone area and reduction in the bone marrow area. These findings can be confirmed in the percentages of the bone showed in Table 1. The osteoporosis group showed a significant decrease in the percentage of the trabecular bone (PTB) and in the percentage of the alveolar bone (PAB) when compared to the control group (Table 1). The animals from the positive control group showed a significant increase in the PTB of the femur. EGb2 and EGb3 also effectively increased the PTB of the femur when compared to the osteoporosis group. The PAB of the mandible had no significant increase in the positive control group, however the groups treated with G. biloba (EGb1, EGb2 and EGb3) showed significant increase in the PAB of the mandible when compared to the osteoporosis group (Table 1). 4. Discussion The studies about the action of glucocorticoids in the jaws are very important because the quantity and quality of the mandibular bone are essential to the planning of prosthetic treatments and implants by dentists [17]. The knowledge about glucocorticoid effects on bone metabolism could help dentists to advise their patients about the risk of systemic and oral bone loss. Olgaard et al. [18] studied bone density of nephropathic patients using corticoids for a prolonged time. They reported a significant decrease of bone mass in many regions of the skeleton, including the mandible. Despite some significant findings, literature is still scant regarding the effects of secondary osteoporosis and the use of glucocorticoids in the
bone tissue of the mandible. Bone histomorphometry is a technique often used to provide information about bone gain as well as bone loss in untreated and treated diseases [19]. Thus the same procedure was used in this study to evaluate the effect of EGb on animals with glucocorticoid-inducedosteoporosis. The PAB of the mandible in the osteoporosis group was significant lower than that of the control group as was demonstrated by previous works [20,21]. The same osteoporotic changes and a thin alveolar bone in the interradicular bone septum were also observed in ovariectomized rats [17], hence the two models of experimental induction of osteoporosis presented the same efficiency. The mechanisms by which glucocorticoids promote the decrease of bone volume are not well characterized; however in vitro studies have suggested that they can have direct effects on the bone-forming osteoblast cells and alter the number of osteoblasts by mechanisms such as the induction of apoptosis [22]. Presently, the best osteoporosis treatment are the biphosphonates, compounds that decrease the osteoclast reabsorptive activity. This decrease alters the structural formation of the osteoclast, thereby causing its cellular death [23]. In the present study, the thirty-day period of intragastric treatment with sodium alendronate (positive control), a second-generation biphosphonate, caused no significant increase in the PAB of the mandible in rats. This datum differs from the results found in the literature [24,25] and this difference could be explained by the time of exposition, since in humans the suppression of bone resorption occurs after three months of daily treatment, and the resorption is suppressed more rapidly in the intravenous treatment when compared to the intragastric administration. The biphosphonate are very hydrophilic to the extent that they are poorly absorbed by the gastrointestinal tract after oral administration. Only 50% of the drug absorbed is retained in the skeleton, the remainder is eliminated by urine without being metabolized [26]. The treatment with EGb at all doses showed a significant increase in the PAB of the mandible when compared to the osteoporosis group. Corroborating this, in a previous study we showed through digital radiographic method that the EGb recovered the periodontal support and increased the mandibular cortical thickness [14]. Thus the effect of EGb seems to be quicker and more effective than that of alendronate in the
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Fig. 2. Frontal sections of the interradicular area of the second molar of the mandible, from control (A), osteoporosis (B), positive control (C), EGb1 (D), EGb2 (E) and EGb3 (F) groups (H&E, original magnification, 25×). Alveolar bone (AB) and marrow bone (BM).
treatment of mandibular osteoporosis. Similarly, the femur of the osteoporotic animals presented a lower PTB when compared to the control group. The positive control group showed a significant increase in the PTB of the femur. This is in accordance with previous studies that reported promising results in the treatment of femur osteoporosis with biphosphonates [15,27].
The effects of EGb on the mandible and femur may be explained by the presence of some flavonoids, such as quercetin and kaempferol, among the constituents of G. Biloba. These two flavonoids have been reported to inhibit osteoclast resorption activity and induce mature osteoclast apoptosis in vitro, indicating a possible anti-resorptive activity [28]. A decrease in the function of osteoclasts is important because bone
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Fig. 3. Longitudinal sections of the epiphiseal area of the femur from control (A), osteoporosis (B), positive control (C), EGb1 (D), EGb2 (E) and EGb3 (F) groups (H&E, 25×). Trabecular bone (TB) and marrow bone (MB).
resorption by these cells is stimulated by the use of glucocorticoids. Kaempferol inhibited the formation of adipocytes in the bone marrow and prevented bone loss induced by ovariectomy in vivo through osteoblastic function promotion and, consequently, increase of osseous neoformation [29].
In vitro studies showed that G. biloba (100 μg/ml) increased alkaline phosphatase levels in 147.2% when compared to the culture of control cells. Similarly, kaempferol and quercetin were also shown to stimulate alkaline phosphatase activity in cultures of human osteoblastic cells
L.M.F. Lucinda et al. / Fitoterapia 81 (2010) 982–987 Table 1 Comparison of the percentage of the PAB and PTB of the mandible and femur. Groups
PTB of the femur (%)
PAB of the mandible (%)
Control Osteoporosis Positive control EGb1 EGb2 EGb3
36.04 ± 0.94 21.05 ± 1.36 ⁎ 32.47 ± 2.26 ⁎⁎
69.90 ± 2.76 51.19 ± 0.89 ⁎ 58.04 ± 5.27 65.66 ± 5.34 ⁎⁎ 73.03 ± 1.07 ⁎⁎ 71.06 ± 1.06 ⁎⁎
26.94 ± 3.06 34.38 ± 4.98 ⁎⁎ 34.45 ± 2.71 ⁎⁎
Values are mean ± S.E.M. ⁎ p b 0.05 when comparing osteoporosis group with the control one (Student's t-test). ⁎⁎ p b 0.05 when comparing all groups, except control, with the osteoporosis group (Dunnett's post-hoc test).
(MG-63) [12,30]. Therefore, EGb action in the alkaline phosphatase may have been responsible for the improvement of the mandibular and femur osteoporosis, since glucocorticoids decreased the levels of this enzyme, a known marker of osteoblastic function and bone mineralization. Moreover, EGb showed estrogenic activity in vitro [8] and this effect is important because estrogen acts on bone cells inhibiting resorption and promoting osteogenesis [31]. In conclusion, the treatment with EGB recovered the bone area of the mandible and femur following induction of osteoporosis with glucocorticoid. This suggests that the extract may be contributing to the treatment of osteoporosis secondary to the use of glucocorticoids, without showing some of the side effects displayed by biphosphonates. Acknowledgments This work was financed by Fundação Mineira de Amparo à Pesquisa (FAPEMIG)-CDS APQ-2069-4.04/07 and Redes Mineiras: 2824/05 and 2827/05. We would like to thank Dr. Roberto Sotto-Maior Fortes de Oliveira for the critical reading of the manuscript. References [1] Dervis E. Oral implications of osteoporosis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;100:349–56. [2] Johnell O. Advences in osteoporosis: better identification of risk factors can reduce morbity and mortality. J Intern Med 1996;239:229–304. [3] Reid IR. Glucocorticoid-induced osteoporosis. Ballière's. Clin Endocrinol Metabol 2000;14:279–98. [4] Szejnfeld. VL. Clinical manifestations. Osteoporosis: diagnostic and treatment. São Paulo: Sarvier; 2000. Portuguese. [5] Pozzi S, Marcheselli R, Sacchi S, Baldini L, Angrilli F, Pennese E, et al. Biphosphonate-associated osteonecrosis of the jaw: a review of 35 cases and an evaluation of its frequence in multiple myeloma patients. Leuk Lynphoma 2007;48:1852–4. [6] Dang GZC, Lowik C. Dose-dependent effects of phytoestrogens on bone. Trends Endrocrinol Metab 2005;16:207–11. [7] Ji YB, Alaerts G, Xu CJ, Hu YZ, Vander Heyden Y. Sequential uniform designs for fingerprints development of Ginkgo biloba extracts by capillary electrophoresis. J Chromatogr A 2006;1128:273–81. [8] Oh SM, Chung KH. Estrogenic activities of Ginkgo biloba extracts. Life Sci 2004;74:1325–35.
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[9] Diamond BJ, Shiflett SC, Feiwel N, Matheis RJ, Noskin O, Richards JA, et al. Ginkgo biloba extract: mechanisms and clinical indications. Arch Phys Med Rehabil 2000;81(2000):668–78. [10] Di Renzo G. Ginkgo biloba and the central nervous system. Fitoterapia 2000;71:43–7. [11] Yamaguchi M, Hamamoto R, Uchyama S, Ishyama K. Effects of flavanoid on calcium content in femoral tissue culture and parathyroid hormonestimulated osteoclastogenesis in bone marrow culture in vitro. Mol Cell Biochem 2007;303:83–8. [12] Oh SM, Kim HR, Chung KH. Effects of Ginkgo biloba on in vitro osteoblasts cells and ovariectomized rat osteoclasts cells. Arch Pharm Res 2008;31:216–24. [13] Brayboy. JR, Chen XW, Lee YS, Anderson JJB. The protective effects of Ginkgo biloba extract (EGb 761) against free radical damage osteoblastlike bone cells (MC3T3-E1) and the proliferative effects of EGb 761 on these cells. Nutr Res 2001;21:1275–85. [14] Lucinda LMF, Oliveira TT, Salvador PA, Peters VM, Reis JEP, Guerra MO. Radiographic evidence of mandibular osteoporosis improvement in Wistar rats treated with Ginkgo biloba. Phytother Res 2010;24:264–7. [15] Pinto A, Oliveira TT, Del Carlo RJ, Nagem TJ, Fonseca CC, Moraes GHK, et al. Effects of combined treatment of sodium alendronate, atorvastatin calcium and ipriflavon in osteoporosis induced in rats with dexamethasone. Rev Bras Cienc Farm 2006;42:99–107 Portuguese. [16] Pinto RM, Fernandes ES, Reis JEP, Peters VM, Guerra MO. Intra-uterine growth retardation after prenatal administration of Ginkgo biloba to rats. Reprod Toxicol 2007;23:480–5. [17] Armada L, Nogueira CRR, Neves UL, Souza PS, Detogne JP, Armada-Dias L, et al. Mandible analysis in sex steroid-deficient rats. Oral Dis 2006;12: 181–6. [18] Olgaard K, Storm T, Van Wowern N, Daugaard H, Egfjord M, Lewin. E, et al. Glucorticoid-induced osteoporosis in the lumbar spine, forearm, and mandible of neprhotic patients: a double-blind study on the highdose, long-term effects of prednisone versus deflazacort. Calcif Tissue Int 1992;50:490–7. [19] Compston J. Bone histomorphometry — the renaissance? BoneKEyosteovision 2004;5:9–12. [20] Fujita Y, Konoo T, Maki K. Short-term treatment prevents glucocorticoid-induce bone debility of the mandible in growing rats. Orthod Craniofac Res 2008;11:187–95. [21] Mahl CR, Fontanella V. Evaluation by digital radiography of induced changes in the bone density of the female rat mandible. Dentomaxillofac Radiol 2008;37:438–44. [22] Silvestini G, Ballanti P, Patacchiioli FR, Mocetti P, Di Grezia R, Wedard BM, et al. Evaluation of apoptosis and the glucocorticoid receptor in the cartilage growth plate and metaphyseal bone cells of rats after highdose treatment with corticosterone. Bone 2000;26:33–42. [23] Hugges DE, Wright KR, Uy HL, Sasaki A, Yoneda T, Roodman GD, et al. Biphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J Bone Miner Res 1995;10:1478–87. [24] Anbinder AL, Prado Fde A, Prado Mde A, Balducci I, da Rocha. RF. The influence of ovariectomy, sinvastatin and sodium alendronate on alveolar bone in rats. Braz Oral Res 2007;21:247–52. [25] El-Shinnawi UM, El-Tantawy SI. The effect of alendronate sodium on alveolar bone loss in periodontitis (clinical trial). J Int Acad Periodontol 2003;5:5–10. [26] Drake MT, Clarke MD, Khosla S. Biphosphonates: mechanism of action and role in clinical practice. Mayo Clin Proc 2008;83:1032–45. [27] Russel RGG. Ibandronate: pharmacology and preclinical studies. Bone 2006;38:7–12. [28] Wattel A, Kamel S, Mentaverri R, Lorget F, Prouillet C, Petit JP, et al. Potent inhibitory effect of naturally occurring flavonoids quercetin and kaempferol on in vitro osteoclastic bone resorption. Biochem Pharmacol 2003;65:35–42. [29] Trivedi R, Kumar S, Kumar A, Siddiqui JA, Swarnkar G, Gupta V, et al. Kaempferol has osteogenic effect in ovariectomized adult Sprague– Dawley rats. Mol Cell Endocrinol 2008;289:85–93. [30] Prouillet C, Mazière J, Mazière C, Wattel A, Brazier M, Kamel S. Stimulatory effect of naturally occurring flavonols quercetin and kaempferol on alkaline phosphatase activity in MG-63 human osteoblasts through ERK and estrogen receptor pathway. Biochem Pharmacol 2004;67:1307–13. [31] Turner RT, Riggs L, Spelsberg TC. Skeletal effects of estrogen. Endocr Rev 1994;15:275–99.
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Evidences of osteoporosis improvement in Wistar rats treated with Ginkgo biloba extract: A histomorphometric study of mandible and femur L.M.F. Lucinda a,⁎, B.J. Vieira a, T.T. Oliveira b, R.C.S. Sá a, V.M. Peters a, J.E.P. Reis a, M.O. Guerra a a b
Centro de Biologia da Reprodução, Federal University of Juiz de Fora, Caixa Postal 328, Zip Code 36001-970. Juiz de Fora, MG, Brazil Departamento de Bioquímica e Biologia Molecular, Federal University of Viçosa, Vila Gianetti, casa 26, Zip Code 36570-000. Viçosa, MG, Brazil
a r t i c l e
i n f o
Article history: Received 23 February 2010 Accepted in revised form 8 June 2010 Available online 25 June 2010 Keywords: Ginkgo biloba Osteoporosis Histomorphometry Sodium alendronate Rats
a b s t r a c t This study was aimed at investigating the anti-osteoporotic effects of the extract of Ginkgo biloba (EGb) in glucocorticoid-induced-osteoporosis. A significant reduction was observed in the percentage of the bone of the osteoporosis group in both the mandible and femur. The EGb group treated with 28 and 56 mg/Kg showed a significant increase in the percentage of trabecular bone (PTB) of the femur. The percentage of the alveolar bone of the mandible (PAB) had a significant increase with all doses of EGb. The treatment with EGb significantly reversed the loss of the PAB of the mandible and of the PTB of the femur. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Osteoporosis is a metabolic bone disease characterized by low bone mass and a microarchitectural deterioration of bone tissue leading to bone fragility and an increase in bone fracture [1]. Low bone mass results from genetic, nutritional and lifestyle factors, decreased estrogen levels and the use of drugs [2], such as glucocorticoids that are widely used in the treatment of chronic diseases because of their immunosuppressive and anti-inflammatory properties. The long-term use of glucocorticoids is associated with numerous side effects, such as the development of osteoporosis. This medication interacts with bone metabolism at many levels, but their principal action is to reduce osteoblast number and bone matrix synthesis. The change in osteoblast activity is followed by a fall in bone mass that is maximal over the first few moments of therapy [3]. Biphosphonates are a group of well documented drugs used in the treatment of osteoporosis, although side effects
⁎ Corresponding author. Rua Doutor Pedro de Aquino Ramos 106/503, Granbery. Zip Code: 36010-440, Juiz de Fora, MG, Brazil. Tel.: + 55 32 21023251, + 55 32 32180734; fax: + 55 32 21023251. E-mail address: [email protected] (L.M.F. Lucinda). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.06.014
like gastrointestinal intolerance [4] and osteonecrosis of the jaws [5] have been reported in some patients. The search for new therapeutic alternatives to treat osteoporosis has been under scrutiny over the years. For instance, studies with phytoestrogens (plant-derived compounds possessing estrogen-like activity) showed that they are promising alternatives to treat osteoporosis [6]. Ginkgo biloba L. (Family Ginkgoaceae) is a native plant from China, Japan and Korea where its fruits and leaves have long been used as a food source and a folk medicine. Phytochemical studies have shown a standard composition of substances in this plant comprising: 24% of phytoestrogens (kaempferol, kercetin and ishorhamnetin), 6% of terpenoids (ginkgolides and bilobilides) and less than 5 ppm of ginkgolic acid [7,8]. The main pharmacological effects of the extract of G. biloba (EGb) are vascular and tissue protection, and improvement in cognitive function [9,10] while the phytoestrogen compounds, such as quercetin, kaempferol and ishorhamnetin, were effective in inhibiting osteoclast activity in vitro [10,11]. A recent study showed that EGb stimulated osteoblast differentiation and antiosteoclastic activity in vitro [12]. These effects together with the antioxidant activity of EGb could be important in the treatment of osteoporosis, since
L.M.F. Lucinda et al. / Fitoterapia 81 (2010) 982–987
this activity protected osteoblasts from cellular damages and death when they were exposed to the action of free radicals in vitro [13]. Our previous study showed by digital radiography that EGb was effective in recovering the periodontal bone support (PBS) and in increasing the cortical thickness of the mandible of rats with glucocorticoid-induced-osteoporosis [14]. Considering the pharmacological effects of G. biloba on bone tissue and cells, and our previous results with digital radiographics [14], the present study was designed to evaluated by histomorphometric technique the effect of the EGb on mandibular and femoral bone of Wistar rats with glicocorticoid-induced-osteoporosis.
983
six groups (n = 6). The control group was not submitted to osteoporosis induction. 1 osteoporosis group (dexamethasone); 2 positive control group (sodium alendronate 0.2 mg/animal/day administered intragastrically, once a day, during 30 days, after the osteoporosis induction) [15]; 3 EGb1 group (G. biloba 14 mg/kg/day); 4 EGb2 group (G. biloba 28 mg/kg/day); 5 EGb3 group (G. biloba 56 mg/kg/day); and 6 control group. The doses of EGb were administered intragastrically, once a day, during 30 days, after the osteoporosis induction. The choice of EGb doses was based on previous studies [16], which suggested an estrogenic effect (intra-uterine growth retardation) in pregnant rats with the dose of 14 mg of EGb/kg/day. The subsequent doses are multiples of the former one.
2. Methods The methodology of this work was approved by the Ethical Committee on Animal Experimentation (protocol number 58/2006, CEEA, Federal University of Juiz de Fora, MG, Brazil), which follows the international principles in ethics for animal experimentation. 2.1. Plant material EGb was supplied by JR Pharma (China-lot no. 06112216c). The quality test made by Galena Laboratory showed that the extract was composed of: 28.2% of ginkgoflavonglicosides; 8.3% of terpenolactones; 15% of quercetin glycosides; 10.9% of kaempferol glycosides; 2.3% of ishorhamnetin glycosides and less than 5 ppm of ginkgolic acids. 2.2. Sodium alendronate The solution of sodium alendronate was supplied by JR Pharma (Solution-lot no SA/ 20060501). 2.3. Animals Female Wistar rats (Rattus norvegicus Berkenhout, 1769) (50 days old and weighing approximately 100–150 g) were obtained from the vivarium of the Federal University of Juiz de Fora (UFJF), where they were born and bred. Groups of three animals were housed in clear plastic cages with stainless steel wire lids and pinewood shavings as bedding and kept in room with controlled environmental conditions (12-h light/12-h dark cycle, temperature 22 °C), on closed ventilated shelves. The animals were fed on rat chow pellets (an average of 25 g daily) and received water ad libitum. 2.4. Osteoporosis induction The osteoporosis induction was done through the intramuscular administration of dexamethasone disodium phosphate (Decadron®-4 mg/ml) at the dose level of 7 mg/kg of body weight, once a week, during five weeks, in all groups, except in the control group [15]. 2.5. Bioassay In this experiment, after the end of osteoporosis induction, 36 animals were selected at random and divided evenly into
2.6. Histomorphometry The samples of the mandible and femur were analyzed in an optical microscope system Zeiss (Hallbergmoos, Germany) for digital capturing photomicrographs. All slides were analyzed in the microscope at 250X and 400× magnifications by a pathologist. Histomorphometric analysis was conducted by only one evaluator, who was unaware of the experimental groups.
2.6.1. Histomorphometric analysis of the femur The trabecular bone tissue of the proximal epiphysis of the femur was photographed at 25× magnification. Three areas were identified: two lateral areas (A and C) and a medial one (B) as shown in Fig. 1. Each region (A, B and C) presented a total area of 16,000 μ2. Through the software Axiovision® (version 4.5 for Windows semi-automatic), the trabecular bone tissue was marked and measured with the assistance of a digital cursor pen. The delineated bone tissue area was converted into proportion of bone tissue according to the following formula: % of bone tissue = area measured ×100/16,000 μ2. After the calculation of the proportion of bone tissue from the regions A, B and C, the mean of the proportions of the femoral epiphysis was established for further statistical processing.
2.6.2. Histomorphometric analysis of the mandible The histomorphometric analysis of the alveolar bone of the mandible was performed in the interradicular septum of the second molar (Fig. 2). The measuring procedure was the same as that used in the femur analysis, with the exception that only the bone tissue limited to the interradicular septum was measured.
2.7. Statistical analysis The data were expressed by mean ± standard error mean (S.E.M.) and were analyzed for statistical significance using one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test. The control group was compared with the osteoporosis group using the Student's t-test. P b 0.05 was considered significant.
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Fig. 1. (A) Schematic drawing of the areas selected for the morphometric analysis of the trabecular bone of the femur (B) and of the interradicular alveolar bone of the mandible.
3. Results Fig. 2 shows the histological sections of the interradicular septum of the second molar in which the reduction of the mandible bone area is identified by a significant reduction of the alveolar bone. The significant reduction of the epiphiseal area of the femur is observed in Fig. 3 and identified by the lower thickness of the trabeculae bone. Both the mandible and femur presented a larger area of bone marrow in the animals with osteoporosis when they were compared to the control group. The treated groups show an increase in the alveolar bone area and reduction of the bone marrow area in the interradicular septum of the second molar. Similarly, the femur shows increase in the trabecular bone area and reduction in the bone marrow area. These findings can be confirmed in the percentages of the bone showed in Table 1. The osteoporosis group showed a significant decrease in the percentage of the trabecular bone (PTB) and in the percentage of the alveolar bone (PAB) when compared to the control group (Table 1). The animals from the positive control group showed a significant increase in the PTB of the femur. EGb2 and EGb3 also effectively increased the PTB of the femur when compared to the osteoporosis group. The PAB of the mandible had no significant increase in the positive control group, however the groups treated with G. biloba (EGb1, EGb2 and EGb3) showed significant increase in the PAB of the mandible when compared to the osteoporosis group (Table 1). 4. Discussion The studies about the action of glucocorticoids in the jaws are very important because the quantity and quality of the mandibular bone are essential to the planning of prosthetic treatments and implants by dentists [17]. The knowledge about glucocorticoid effects on bone metabolism could help dentists to advise their patients about the risk of systemic and oral bone loss. Olgaard et al. [18] studied bone density of nephropathic patients using corticoids for a prolonged time. They reported a significant decrease of bone mass in many regions of the skeleton, including the mandible. Despite some significant findings, literature is still scant regarding the effects of secondary osteoporosis and the use of glucocorticoids in the
bone tissue of the mandible. Bone histomorphometry is a technique often used to provide information about bone gain as well as bone loss in untreated and treated diseases [19]. Thus the same procedure was used in this study to evaluate the effect of EGb on animals with glucocorticoid-inducedosteoporosis. The PAB of the mandible in the osteoporosis group was significant lower than that of the control group as was demonstrated by previous works [20,21]. The same osteoporotic changes and a thin alveolar bone in the interradicular bone septum were also observed in ovariectomized rats [17], hence the two models of experimental induction of osteoporosis presented the same efficiency. The mechanisms by which glucocorticoids promote the decrease of bone volume are not well characterized; however in vitro studies have suggested that they can have direct effects on the bone-forming osteoblast cells and alter the number of osteoblasts by mechanisms such as the induction of apoptosis [22]. Presently, the best osteoporosis treatment are the biphosphonates, compounds that decrease the osteoclast reabsorptive activity. This decrease alters the structural formation of the osteoclast, thereby causing its cellular death [23]. In the present study, the thirty-day period of intragastric treatment with sodium alendronate (positive control), a second-generation biphosphonate, caused no significant increase in the PAB of the mandible in rats. This datum differs from the results found in the literature [24,25] and this difference could be explained by the time of exposition, since in humans the suppression of bone resorption occurs after three months of daily treatment, and the resorption is suppressed more rapidly in the intravenous treatment when compared to the intragastric administration. The biphosphonate are very hydrophilic to the extent that they are poorly absorbed by the gastrointestinal tract after oral administration. Only 50% of the drug absorbed is retained in the skeleton, the remainder is eliminated by urine without being metabolized [26]. The treatment with EGb at all doses showed a significant increase in the PAB of the mandible when compared to the osteoporosis group. Corroborating this, in a previous study we showed through digital radiographic method that the EGb recovered the periodontal support and increased the mandibular cortical thickness [14]. Thus the effect of EGb seems to be quicker and more effective than that of alendronate in the
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Fig. 2. Frontal sections of the interradicular area of the second molar of the mandible, from control (A), osteoporosis (B), positive control (C), EGb1 (D), EGb2 (E) and EGb3 (F) groups (H&E, original magnification, 25×). Alveolar bone (AB) and marrow bone (BM).
treatment of mandibular osteoporosis. Similarly, the femur of the osteoporotic animals presented a lower PTB when compared to the control group. The positive control group showed a significant increase in the PTB of the femur. This is in accordance with previous studies that reported promising results in the treatment of femur osteoporosis with biphosphonates [15,27].
The effects of EGb on the mandible and femur may be explained by the presence of some flavonoids, such as quercetin and kaempferol, among the constituents of G. Biloba. These two flavonoids have been reported to inhibit osteoclast resorption activity and induce mature osteoclast apoptosis in vitro, indicating a possible anti-resorptive activity [28]. A decrease in the function of osteoclasts is important because bone
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Fig. 3. Longitudinal sections of the epiphiseal area of the femur from control (A), osteoporosis (B), positive control (C), EGb1 (D), EGb2 (E) and EGb3 (F) groups (H&E, 25×). Trabecular bone (TB) and marrow bone (MB).
resorption by these cells is stimulated by the use of glucocorticoids. Kaempferol inhibited the formation of adipocytes in the bone marrow and prevented bone loss induced by ovariectomy in vivo through osteoblastic function promotion and, consequently, increase of osseous neoformation [29].
In vitro studies showed that G. biloba (100 μg/ml) increased alkaline phosphatase levels in 147.2% when compared to the culture of control cells. Similarly, kaempferol and quercetin were also shown to stimulate alkaline phosphatase activity in cultures of human osteoblastic cells
L.M.F. Lucinda et al. / Fitoterapia 81 (2010) 982–987 Table 1 Comparison of the percentage of the PAB and PTB of the mandible and femur. Groups
PTB of the femur (%)
PAB of the mandible (%)
Control Osteoporosis Positive control EGb1 EGb2 EGb3
36.04 ± 0.94 21.05 ± 1.36 ⁎ 32.47 ± 2.26 ⁎⁎
69.90 ± 2.76 51.19 ± 0.89 ⁎ 58.04 ± 5.27 65.66 ± 5.34 ⁎⁎ 73.03 ± 1.07 ⁎⁎ 71.06 ± 1.06 ⁎⁎
26.94 ± 3.06 34.38 ± 4.98 ⁎⁎ 34.45 ± 2.71 ⁎⁎
Values are mean ± S.E.M. ⁎ p b 0.05 when comparing osteoporosis group with the control one (Student's t-test). ⁎⁎ p b 0.05 when comparing all groups, except control, with the osteoporosis group (Dunnett's post-hoc test).
(MG-63) [12,30]. Therefore, EGb action in the alkaline phosphatase may have been responsible for the improvement of the mandibular and femur osteoporosis, since glucocorticoids decreased the levels of this enzyme, a known marker of osteoblastic function and bone mineralization. Moreover, EGb showed estrogenic activity in vitro [8] and this effect is important because estrogen acts on bone cells inhibiting resorption and promoting osteogenesis [31]. In conclusion, the treatment with EGB recovered the bone area of the mandible and femur following induction of osteoporosis with glucocorticoid. This suggests that the extract may be contributing to the treatment of osteoporosis secondary to the use of glucocorticoids, without showing some of the side effects displayed by biphosphonates. Acknowledgments This work was financed by Fundação Mineira de Amparo à Pesquisa (FAPEMIG)-CDS APQ-2069-4.04/07 and Redes Mineiras: 2824/05 and 2827/05. We would like to thank Dr. Roberto Sotto-Maior Fortes de Oliveira for the critical reading of the manuscript. References [1] Dervis E. Oral implications of osteoporosis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;100:349–56. [2] Johnell O. Advences in osteoporosis: better identification of risk factors can reduce morbity and mortality. J Intern Med 1996;239:229–304. [3] Reid IR. Glucocorticoid-induced osteoporosis. Ballière's. Clin Endocrinol Metabol 2000;14:279–98. [4] Szejnfeld. VL. Clinical manifestations. Osteoporosis: diagnostic and treatment. São Paulo: Sarvier; 2000. Portuguese. [5] Pozzi S, Marcheselli R, Sacchi S, Baldini L, Angrilli F, Pennese E, et al. Biphosphonate-associated osteonecrosis of the jaw: a review of 35 cases and an evaluation of its frequence in multiple myeloma patients. Leuk Lynphoma 2007;48:1852–4. [6] Dang GZC, Lowik C. Dose-dependent effects of phytoestrogens on bone. Trends Endrocrinol Metab 2005;16:207–11. [7] Ji YB, Alaerts G, Xu CJ, Hu YZ, Vander Heyden Y. Sequential uniform designs for fingerprints development of Ginkgo biloba extracts by capillary electrophoresis. J Chromatogr A 2006;1128:273–81. [8] Oh SM, Chung KH. Estrogenic activities of Ginkgo biloba extracts. Life Sci 2004;74:1325–35.
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