Osteoprotective effects of Spilanthes acmella ethanolic leaf extract on bone histomorphometry, biomechanics, and microarchitecture through antioxidant mechanisms in ovariectomized rats

Osteoporosis is a metabolic disease characterized by reduced bone mineral density, leading to an increased risk of fractures. Spilanthes acmella (SA) has demonstrated notable antioxidant activity, attributed to its phenolic and flavonoid compounds. This study aimed to evaluate the osteoprotective effects of an ethanolic leaf extract of SA in an ovariectomized rat model of osteoporosis. A total of 42 female Sprague-Dawley rats were used in this study, comprising 18 in the preliminary phase and 24 in the main intervention. To determine the optimal dosage, the preliminary phase was equally allocated into six groups: sham-operated (Sham), ovariectomized control (OVX), OVX treated with 64.5 µg/kg Premarin (OVX+E), and three OVX groups treated with SA ethanolic leaf extract at doses of 25 mg/kg (OVX+SA25), 50 mg/kg (OVX+SA50), and 100 mg/kg (OVX+SA100). Upon identifying 100 mg/kg as the optimum dose, the intervention phase was divided into four equal groups: Sham, OVX, OVX+E, and OVX treated with 100 mg/kg SA ethanolic leaf extract (OVX+SA). Daily oral treatments were administered for 12 weeks. Blood serum samples were collected before and after treatment to measure bone biochemical markers. After 12 weeks, all rats were sacrificed. The left femurs were harvested for bone histomorphometry analysis (structural, dynamic, and static), while the right femurs were assessed for biomechanical strength and antioxidant enzyme activities. The left tibiae were analyzed for bone microarchitecture. Structural histomorphometry showed that both the OVX+E and OVX+SA groups had significantly better trabecular bone volume, number, and separation compared to the OVX group (p < 0.05). Dynamic histomorphometry revealed significantly increased double-labeled surface, mineralizing surface, mineral appositional rate, and bone formation rate in the OVX+SA group compared to the Sham and OVX groups (p < 0.05). Static histomorphometry indicated that the percentages of osteoblast and osteoid surfaces were significantly higher in the OVX+E and OVX+SA groups compared to the OVX group (p < 0.05), whereas the percentages of osteoclast and eroded surfaces were significantly lower. Biomechanical strength analysis showed that maximum stress and Young’s modulus were significantly higher in the OVX+E and OVX+SA groups than in the untreated groups (p < 0.05). Microarchitectural analysis of the tibial trabecular bone revealed that trabecular bone volume, thickness, number, and connectivity density values in the OVX, OVX+E, and OVX+SA groups were significantly lower than in the Sham group (p < 0.05). However, the cortical area and thickness values of tibial cortical bone in the OVX+SA group were significantly higher compared to the Sham group (p < 0.05). Biochemical analysis showed that osteoprotegerin and osteocalcin levels were elevated in the OVX+SA group after treatment (p < 0.05), whereas the OVX group exhibited reduced superoxide dismutase and glutathione peroxidase levels and increased malondialdehyde levels. Supplementation with SA and Premarin reversed these effects, as shown by increased superoxide dismutase and glutathione peroxidase activities and decreased malondialdehyde levels in the OVX+E and OVX+SA groups (p < 0.05). This study is the first to determine the combined antioxidant and osteogenic mechanisms of Spilanthes acmella in an estrogen-deficient model, providing strong preclinical evidence by supporting this plant as a natural alternative to conventional hormone replacement therapies in managing postmenopausal osteoporosis. In conclusion, supplementation with 100 mg/kg of SA demonstrated potential osteoprotective effects, capable of restoring bone loss induced by ovariectomy.