Stereological investigation of the effect of Elaeagnus angustifolia fruit hydroalcoholic extract on osteoporosis in ovariectomized rats

Document Type: Original Research Article


1 Department of Endocrinology, Endocrinology and Metabolism Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

2 Department of Anatomy, Histomorphometry and Stereology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

3 Department of pharmacology, Stem Cell and Transgenic Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

4 Researcher in Central Research Laboratory, Shiraz University of Medical Sciences, Shiraz, Iran

5 Researcher in Health Policy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran


Objective: Postmenopausal osteoporosis is characterized by increased fracture risk. However, each approved treatment has specific side effects. Therefore, foods with plant origins have increasingly attracted attention as an alternative treatment. Studies have shown that Elaeagnus angustifolia (EA) has antioxidant properties. The present study aimed to investigate the effects of EA hydroalcoholic extract on ovariectomy-induced osteoporosis in rats using stereological methods. 
Material and Methods: 55 female Sprague-Dawley rats were randomly assigned to control, sham operated (normal saline), ovariectomized (OVX), OVX + EA fruit extract (600 mg/kg BW/day), and OVX + estradiol benzoate (3 mg/kg BW) for 16 weeks. Blood samples were collected to measure calcium, phosphorus, and alkaline phosphatase (ALP) plasma levels. Then, specimens from tibia and fifth lumbar vertebra (L5) bones were prepared and stereological analysis was done.
Results: Ovariectomy significantly decreased the calcium level and increased the ALP level in the OVX group. In spite of improvement in calcium hemostasis in groups treated with estrogen and EA fruit extract (p<0.05), only treatment with estrogen was able to reduce ALP levels. Moreover, treatment with EA fruit extract and estrogen caused a significant increase in the number of osteoblasts in vertebra and tibia compared to the OVX group (p<0.05). Estrogen and EA fruit extract were also able to reduce the number of osteoclasts in tibia of the treated OVX rats (p<0.05).
Conclusion: The results showed that EA extract exerted more effects, markedly, on osteoblastogenesis in the OVX rats. Thus, it could be considered as a potential agent to treat patients with osteoporosis.


Main Subjects

Ashouri E, Meimandi EM, Saki F, Dabbaghmanesh MH, Omrani GR, Bakhshayeshkaram M. 2015. The impact of LRP5 polymorphism (rs556442) on calcium homeostasis, bone mineral density, and body composition in Iranian children. J Bone Miner Metab, 33:651-657
Bekker NP, Glushenkova AI. 1997. Lipids of the leaves of Elaeagnusangustifolia. Chem Nat Compd, 33: 543–544.
Bidwell JP, Alvarez MB, Hood M Jr, Childress P. 2013. Functional impairment of bone formation in the pathogenesis of osteoporosis: the bone marrow regenerative competence. Curr Osteoporos Rep, 11:117-125.
Bucur L, Negreanu-Pîrjol T, Giurginca M, Istudor V. 2008. Some new Elaeagnus angustifolia l. Extracts and the pharmaceutical products` antioxidant activities determined by the chemiluminiscence method. Revue. Roumaine. Du Chimie, 53: 961-964.
Callaway DA, Jiang JX. 2015. Reactive oxygen species and oxidative stress in osteoclastogenesis, skeletal aging and bone diseases. J Bone Miner Metab, 33: 359-370.
Chen JR, Lazarenko OP, Wu X, Kang J, Blackburn ML, Shankar K, Badger TM, Ronis MJ. 2010. Dietary-induced serum phenolic acids promote bone growth via p38 MAPK/β-catenin canonical Wnt signaling. J Bone Miner Res, 25: 2399-2411.
Chen Q, Chen J, Du H, Li Q, Chen J, Zhang G, Liu H, Wang J. 2014. Structural characterization and antioxidant activities of polysaccharides extracted from the pulp of Elaeagnus angustifolia L. Int J Mol Sci, 26: 15: 11446-11455.
Choi, HJ. 2015. New antiresorptive therapies for postmenopausal osteoporosis. J Menopausal Med, 21: 1-11.
D’Amelio P, Roato I, D’Amico L, Veneziano L, Suman E, Sassi F,Bisignano G, Ferracini R, Gargiulo G, Castoldi F, Pescarmona GP,Isaia GC. 2011. Bone and bone marrow pro-osteoclastogenic cytokines are up regulated in osteoporosis fragility fractures. Osteoporos Int, 22: 2869-2877.
D’Amelio, Sr. FS. Botanicals. CRC Press LLC. USA. 1999, pp: 39 - 41.
Ebrahimi AA, Nikniaz Z, Ostadrahimi A, Mahdavi R, Nikniaz L. 2014. "The effect of Elaeagnus angustifolia L. whole fruit and medulla powder on women with osteoarthritis of the knee: A randomized controlled clinical trial. Eur J Integ Med, 6: 672-679.
Farzaei MH, Bahramsoltani R, Abbasabadi Z, Rahimi RA. 2015. Comprehensive review on phytochemical and pharmacological aspects of Elaeagnus angustifolia L.J Pharm Pharmacol, 67: 1467-1480.
Farrier AJ, Sanchez Franco LC, Shoaib A, Gulati V, Johnson N, Uzoigwe CE, Choudhury MZ. 2016. New anti-resorptives and antibody mediated anti-resorptive therapy. Bone Joint J, 98-B:160-165.
Garnero P. 2014.  New developments in biological markers of bone metabolism in osteoporosis. Bone, 66: 46-55
 García-Villalba R, Larrosa M, Possemiers S, Tomás-Barberán FA, EspínJC. 2014. Bioavailabilityof phenolics from an oleuropein-rich olive (Oleaeuropaea) leaf extract and its acute effect on plasma antioxidant status: comparison between pre-and postmenopausal women. Eur J Nutr, 53: 1015-1027.
Gatta LA, Jiang X, Schnatz PF. 2015.  Hormone therapy in women with primary ovarian insufficiency or early menopause. Menopause, 22: 923-925.
 Goldring SR. 2015. Inflammatory signaling induced bone loss. Bone, 80:143-149
 Greenwald P, Anderson D, Nelson SA, Taylor PR. 2007.  Clinical trials of vitamin andmineral supplements for cancer prevention. Am J clinNutr, 85: 314S-317S.
 Greenspan SL, Bone HG, Ettinger MP, Hanley DA, Lindsay R,Zanchetta JR, Blosch CM, Mathisen AL, Morris SA, Marriott TB. 2007. Effect of recombinant human parathyroid hormone (1–84) on vertebral fracture and bone mineral density in postmenopausalwomen with osteoporosis: a randomized trial. Ann Intern Med, 146: 326–339
Gundersen HJ, Bendtsen TF, Korbo L, Marcussen N, Møller A, Nielsen K, Nyengaard JR, Pakkenberg B, Sørensen FB, Vesterby A. 1988. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS. 96: 379- 394.
 Hohman EE, Weaver CM. 2015. A grape-enriched diet increases bone calcium retention and cortical bone properties in ovariectomized rats. J Nutr, 145: 253-259.
 Jeddi M, Roosta MJ, Dabbaghmanesh MH, Omrani GR, Ayatollahi SM, Bagheri Z, Showraki AR, Bakhshayeshkaram M. 2013. Normative data and percentile curves of bone mineral density in healthy Iranian children aged 9-18 years. Arch Osteoporos, 8: 114.
 Jia M, Nie Y, Cao DP, Xue YY, Wang JS, Zhao L, Rahman K, Zhang QY, Qin LP. Potential antiosteoporotic agents from plants: a comprehensive review. 2012. Evid Based Complement Alternat Med, 2012:1-29.
Karbalay-Doust S, Noorafshan A, Pourshahid SM. 2012. Taxol and Taurine Protect the Renal Tissue of Rats after Unilateral Ureteral Obstruction: A Stereological Survey. Korean J Urol, 53: 360–367.
Khodakarm-Tafti A, Mehrabani D, Homafar L, Farjanikish G. 2015. Healing Effects of Elaeagnus angustifolia Extract in Experimentally Induced Ulcerative Colitis in Rats. J Pharmacol Toxicol, 10: 29-35.
Khosla S, Oursler MJ, Monroe DG. 2012. Estrogen and the skeleton. Trends Endocrinol Metab, 23: 576-581
 Kim OY, Chae JS, Paik JK, Seo HS, Jang Y, Cavaillon JM, Lee JH. 2012. Effects of aging and menopause on serum interleukin-6 levels and peripheral blood mononuclear cell cytokine production in healthy nonobese women. Age (Dordr), 34: 415-425.
Mattfeldt T, Mall G, Gharehbaghi H, Möller P. 1990.  Estimation of surface area and length with the orientator. J Microsc, 159: 301-17
Manolagas SC. 2010. from estrogencentric to aging and oxidative stress: a revised perspective of the pathogenesis of osteoporosis. Endocr Rev, 31: 266-300.
 Mok SK, Chen WF, Lai WP, Leung PC, Wang XL, Yao XS, Wong MS. 2010 Icariin protects against bone loss induced by oestrogen deficiency and activates oestrogen receptor-dependent osteoblastic functions in UMR 106 cells.Br J Pharmacol, 159: 939-949.
Mukaiyama K, Kamimura M, Uchiyama S, Ikegami S, Nakamura Y, Kato H. 2015. Elevation of serum alkaline phosphatase (ALP) level in postmenopausal women is caused by high bone turnover. Aging Clin Exp Res, 27: 413-8.
Nikniaz Z, Ostadrahimi A, Mahdavi R, Ebrahimi AA, Nikniaz L. 2014. Effects of Elaeagnus angustifolia L. supplementation on serum levels of inflammatory cytokines and matrix metalloproteinases in females with knee osteoarthritis. Complement Ther Med, 22: 864-9.
Nishide Y, Tadaishi M, Kobori M, Tousen Y, Kato M, Inada M, Miyaura C, Ishimi Y. 2013. Possible role of S-equol on bone loss via amelioration of inflammatory indices in ovariectomizedmice. J Clin Biochem Nutr 53:41–8
 Noorafshan A, Dabbaghmanesh MH, Tanideh N, Koohpeyma F, Rasooli R, Hajihoseini M, Bakhshayeshkaram M, Hosseinabadi OK. 2015. Stereological study of the effect of black olive hydroalcoholic extract on osteoporosis in vertebra and tibia in ovariectomized rat. Osteoporos Int, 26: 2299-2307
Noorafshan A, Hoseini L, Karbalay-Doust1 S, Nadimi E. 2012.  A Simple Stereological Method for Estimating the Number and the Volume of the Pancreatic Beta Cells. JOP, 10: 13: 427-432. 
Noorafshan ADabbaghmanesh MHTanideh NKoohpeyma FRasooli RHajihoseini MBakhshayeshkaram MHosseinabadi OK. 2015.  Stereological study of the effect of black olive hydroalcoholic extract on osteoporosis in vertebra and tibia in ovariectomized rat. Osteoporos Int, 26: 2299-2307.
 Nyengaard JR. 1999. Stereologic methods and their application in kidney research. J Am Soc Nephrol, 10: 1100-23.
Peel N. 2009. Bone remodelling and disorders of bone metabolism. Surgery, 27: 470– 482.
Puel C, Quintin A, Agalias A, Mathey J, Obled C, Mazur A, Davicco MJ, Lebecque P, Skaltsounis AL, Coxam V . 2004. Olive oil and its main phenolic micronutrient (oleuropein) prevent inflammation-induced bone loss in the ovariectomised rat. Br J Nutr, 92: 119–127
Saleh NKSaleh HA. 2011. Olive Oil effectively mitigates ovariectomy-induced osteoporosis in rats. BMC Complement Altern Med, 4; 11:10
 Sánchez-Rodríguez MA, Ruiz-Ramos M, Correa-Muñoz E, Mendoza- Núñez VM. 2007. Oxidative stress as a risk factor for osteoporosis in elderly Mexicans as characterized by antioxidant enzymes. BMC Musculoskelet Disord, 19; 8:124.
 Si CL, Qin PP, Lu YY, Wu L, Wang HH, HuiLF, LiuZ, NiYH. 2011.  GC-MS analysis of chemical composition and free radical scavenging activity of Elaeagnus angustifolia bark. Adv Mater Res, 183–185: 854–858. 
 Waterhouse AL, “Determination of total phenolics,” in Current Protocols in Food Analytical Chemistry, R. E. Wrolstad, Ed., units I, pp. I1.1.1–I1.1.8, John Wiley & Sons, New York, NY, USA, 2002.
   Wang Y, Guo T, Li J, Zhou S, Zhao P, Fan M. 2013. Four flavonoidglycosides from the pulps of Elaeagnus angustifolia and theirantioxidant activities. Adv Mater Res, 756-759: 16-20.
Xufeng He and Qiang Shen Salvianolic acid B promotes bone formation by increasing activity of alkaline phosphatase in a rat tibia fracture model: a pilot study
Zargari A, editor. Medicinal plants. Tehran: Tehran University Press; 1990