Bone Fracture-healing Properties and UPLC-MS Analysis of an Enriched Flavonoid Fraction from Oxystelma esculentum

Abstract

Oxystelma esculentum has been used as a folk medicine to treat jaundice, throat infections, and skin problems. In the current study, the bone fracture-healing properties of a flavonoid-enriched fraction (Oxy50-60F) of O. esculentum were investigated in Swiss mice using a drill-hole injury model. Oxy50-60F (1 mg/kg/day, 5 mg/kg/day, and 10 mg/kg/day) was administered orally (from the next day) after a 0.6 mm drill-hole injury in mice femur mid-diaphysis for 7 days and 14 days. Parathyroid hormone (40 µg/kg; 5 times/week) was given subcutaneously as the positive control. Confocal imaging for bone regeneration, micro-architecture of femur bones, ex vivo mineralization, hematoxyline and eosin staining, measurement of reactive oxygen species, and gene expression of osteogenic and anti-inflammatory genes were studied. Quercetin, kaempferol, and isorhamnetin glycosides were identified in the active fraction using mass spectrometry techniques. Our results confirm that Oxy50-60F treatment promotes fracture healing and callus formation at drill-hole sites and stimulates osteogenic and anti-inflammatory genes. Oxy50-60F administration to fractured mice exhibited significantly better micro-CT parameters in a dose-dependent manner and promoted nodule mineralization at days 7 and 14 post-injury. Oxy50-60F also prevents ROS generation by increasing expression of the SOD2 enzyme. Overall, this study reveals that Oxy50-60F has bone regeneration potential in a cortical bone defect model, which supports its use in delayed-union and non-union fracture cases.

Key words

Oxystelma esculentum - Asclepiadaceae - flavonoids - fracture healing - osteoblast differentiation - drill-hole injury

Publication History

Received: 25 September 2022

Accepted after revision: 16 October 2023

Accepted Manuscript online:
16 October 2023

Article published online:
10 November 2023

• References

• 1 Pandya DJ, Anand IS. A complete review on Oxystelma esculentum R. Br. Pharmacogn J 2011; 3: 87-90
• 2 Kirtikar KR, Basu BD. Indian Medicinal Plants, Vol. III. New Delhi: Periodical Experts; 1975
• 3 Watt G. Dictionary of Economic Products of India, Vol. V. Delhi: Periodical Experts; 1891
• 4 Pandya DJ, Anand IS. Anti-ulcer potential of Oxystelma esculentum . Int J Green Pharm (IJGP) 2011; 5: 65-68
• 5 Hamed AI, Sheded MG, Shaheen Ael-S, Hamada FA, Pizza C, Piacente S. Polyhydroxypregnane glycosides from Oxystelma esculentum var. alpine. Phytochemistry 2011; 65: 975-980
• 6 Panche AN, Diwan AD, Chandra SR. Flavonoids: An overview. J Nutr Sci 2016; 5: e47-e47
• 7 Welch AA, Hardcastle AC. The effects of flavonoids on bone. Curr Osteoporos Rep 2004; 12: 205-210
• 8 Weaver CM, Alekel DL, Ward WE, Ronis MJ. Flavonoid intake and bone health. J Nutr Gerontol Geriatr 2012; 31: 239-253
• 9 McCauley J, Bitsaktsis C, Cottrell J. Macrophage subtype and cytokine expression characterization during the acute inflammatory phase of mouse bone fracture repair. J Orthop Res 2020; 38: 1693-1702
• 10 Marsell R, Einhorn TA. The biology of fracture healing. Injury 2011; 42: 551-555
• 11 Dimitriou R, Tsiridis E, Giannoudis PV. Current concepts of molecular aspects of bone healing. Injury 2006; 36: 1392-1404
• 12 Wong RMY, Wong PY, Liu C, Wong HW, Chung YL, Chow SKH, Law SW, Cheung WH. The imminent risk of a fracture-existing worldwide data: a systematic review and meta-analysis. Osteoporos Int 2022; 33: 2453-2466
• 13 Ström O, Borgström F, Kanis JA, Compston J, Cooper C, McCloskey EV, Jönsson B. Osteoporosis: Burden, health care provision and opportunities in the EU: a report prepared in collaboration with the International Osteoporosis Foundation (IOF) and the European Federation of Pharmaceutical Industry Associations (EFPIA). Arch Osteoporos 2011; 6: 59-155
• 14 Hernlund E, Svedbom A, Ivergård M, Compston J, Cooper C, Stenmark J, McCloskey EV, Jönsson B, Kanis JA. Osteoporosis in the European Union: Medical management, epidemiology and economic burden. A report prepared in collaboration with the International Osteoporosis Foundation (IOF) and the European Federation of Pharmaceutical Industry Associations (EFPIA). Arch Osteoporos 2013; 8: 1-115
• 15 Kanis JA, Cooper C, Rizzoli R, Abrahamsen B, Al-Daghri NM, Brandi ML, Cannata-Andia J, Cortet B, Dimai HP, Ferrari S, Hadji P, Harvey NC, Kraenzlin M, Kurth A, McCloskey E, Minisola S, Thomas T, Reginster JY. Identification and management of patients at increased risk of osteoporotic fracture: Outcomes of an ESCEO expert consensus meeting. Osteoporos Int 2017; 28: 2023-2034
• 16 Ahmad N, Chillara R, Karvande A, Tripathi AK, Kothari P, Kushwaha P, Adhikary S, Maurya R, Trivedi R. Ethyl acetate fraction from Passiflora foetida promotes rat femoral fracture healing by the BMP-2 signaling pathway. Int J Regen Med 2018; 14: 1-3
• 17 ElKhateeb A, Hussein S, Salem M, El Negoumy S. LC-ESI-MS Analysis, Antitumor and antiviral activities of Bosica senegalensis aqueous methanolic extract. Egypt J Chem 2019; 62: 77-83
• 18 Li D, Schmitz OJ. Comprehensive two-dimensional liquid chromatography tandem diode array detector (DAD) and accurate mass QTOF-MS for the analysis of flavonoids and iridoid glycosides in Hedyotis diffusa . Anal Bioanal Chem 2015; 407: 231-240
• 19 Duong PQ, Duyen NT, Quyen PT, Tung NQ, Son VH, Hung VD. Isolation and identification of phenolic compounds from the leaf extract of Cassia alata L. Vietnam J Chem 2017; 55: 589-594
• 20 Kazuma K, Noda N, Suzuki M. Malonylated flavonol glycosides from the petals of Clitoria ternatea . Phytochemistry 2023; 62: 229-237
• 21 Wolfram K, Schmidt J, Wray V, Milkowski C, Schliemann W, Strack D. Profiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus). Phytochemistry 2010; 71: 1076-1084
• 22 Yoshida T, Saito T, Kadoya S. New acylated flavonol glucosides in Allium tuberosum Rottler. Chem Pharm Bull 1987; 35: 97-107
• 23 Tang J, Xie J, Chen W, Tang C, Wu J, Wang Y, Zhou XD, Zhou HD, Li YP. Runt-related transcription factor 1 is required for murine osteoblast differentiation and bone formation. J Biol Chem 2020; 295: 11669-11681
• 24 Mizuno M, Fujisawa R, Kuboki Y. Type I collagen-induced osteoblastic differentiation of bone-marrow cells mediated by collagen-α2β1 integrin interaction. J Cell Physiol 2000; 184: 207-213
• 25 Cao L, Wang J, Zhang Y, Tian F, Wang C. Osteoprotective effects of flavonoids: Evidence from in-vivo and in vitro studies. Mol Med Rep 2022; 25: 1-9
• 26 Trivedi R, Kumar S, Kumar A, Siddiqui JA, Swarnkar G, Gupta V, Kendurker A, Dwivedi AK, Romero JR, Chattopadhyay N. Kaempferol has osteogenic effect in ovariectomized adult Sprague–Dawley rats. Mol Cell Endocrinol 2008; 289: 85-93
• 27 Nguyen LT, Maidar O, Sharma AR, Sharma G, Kim HC, Park S, Lee SS, Nam JS. Enhanced healing process of fracture by treatment of kaempferol. Biomed Res 2016; 27: 138-144
• 28 Kim YJ, Bae YC, Suh KT, Jung JS. Quercetin, a flavonoid, inhibits proliferation and increases osteogenic differentiation in human adipose stromal cells. Biochem Pharmacol 2006; 72: 1268-1278
• 29 Yang L, Chen Q, Wang F, Zhang G. Antiosteoporotic compounds from seeds of Cuscuta chinensis. J Ethnopharmacol 2011; 135: 553-560
• 30 Holzer G, Majeska RJ, Lundy MW, Hartke JR, Einhorn TA. Parathyroid hormone enhances fracture healing. A preliminary report. Clin Orthop Relat Res 1999; 366: 258-263
• 31 Lorenzo J. Interactions between immune and bone cells: new insights with many remaining questions. J Clin Invest 2000; 106: 749-752
• 32 Karvande A, Khedgikar V, Kushwaha P, Ahmad N, Kothari P, Verma A, Kumar P, Nagar GK, Mishra PR, Maurya R, Trivedi R. Heartwood extract from Dalbergia sissoo promotes fracture healing and its application in ovariectomy-induced osteoporotic rats. J Pharm Pharmacol 2017; 69: 1381-1397
• 33 Prakash R, Mishra T, Dev K, Sharma K, Kuldeep J, John AA, Tripathi A, Sharma C, Arya KR, Kumar B, Siddiqi MI, Tadigoppula N, Singh D. Phenanthrenoid coelogin isolated from coelogyne cristata exerts osteoprotective effect through MAPK-mitogen-activated protein kinase signaling pathway. Calcif Tissue Int 2021; 109: 32-43
• 34 Pal S, Kumar P, Ramakrishna E, Kumar S, Porwal K, Kumar B, Arya KR, Maurya R, Chattopadhyay N. Extract and fraction of Cassia occidentalis L. (a synonym of Senna occidentalis) have osteogenic effect and prevent glucocorticoid-induced osteopenia. J Ethnopharmacol 2019; 235: 8-18
• 35 Dixit M, Raghuvanshi A, Gupta CP, Kureel J, Mansoori MN, Shukla P, John AA, Singh K, Purohit D, Awasthi P, Singh D, Goel A. Medicarpin, a natural pterocarpan, heals cortical bone defect by activation of notch and wnt canonical signaling pathways. PLoS One 2015; 10: e0144541
• 36 Pal S, Porwal K, Singh H, Malik MY, Rashid M, Kulkarni C, Khan Y, Jagavelu K, Wahajuddin M, Chattopadhyay N. Reversal of osteopenia in ovariectomized rats by pentoxifylline: evidence of osteogenic and osteo-angiogenic roles of the drug. Calcif Tissue Int 2019; 105: 294-307
• 37 Mansoori MN, Shukla P, Kakaji M, Tyagi AM, Srivastava K, Shukla M, Dixit M, Kureel J, Gupta S, Singh D. IL-18BP is decreased in osteoporotic women: Prevents Inflammasome mediated IL-18 activation and reduces Th17 differentiation. Sci Rep 2016; 6: 33680
• 38 Meng XM, Ren GL, Gao L, Yang Q, Li HD, Wu WF, Huang C, Zhang L, Lv XW, Li J. NADPH oxidase 4 promotes cisplatin-induced acute kidney injury via ROS-mediated programmed cell death and inflammation. Lab Invest 2018; 98: 63-78