| Osteoporosis is a disease characterized by low bone mass and a high risk of fracture.Due to its high incidence and long treatment costs,osteoporosis imposes huge economic burden to patients and society.Moreover,common anti-osteoporosis drugs have obvious side effects.It is critical to excavate effective natural plants and functional components to improve osteoporosis.Clinacanthus Nutans is a medicinal homologue used to treat fractures,injuries,swelling and pain.Active components of Clinacanthus nutans leaves such as flavonoids,saponins and polysaccharides have potential effects on improving osteoporosis.Our study firstly explored the effect of crude extract from Clinacanthus nutans leaves in vivo,and further purified flavonoids,saponins and polysaccharides from the crude extract.We screened out saponins was the main substances that affecting bone metabolism.Furthermore,the molecular mechanism of saponins on osteogenic function was explored by using osteoblasts in vitro.Finally,the extraction process of total saponins from Clinacanthus nutans was optimized.The results provide theoretical basis for the pharmacological action of Clinacanthus nutans leaves in improving osteoporosis.The main research contents and results are as follows:Firstly,adult female rats were ovariectomized and osteoporosis model was established to investigate the effect of alcohol and water extracts from Clinacanthus nutans leaves on osteoporosis.The results showed that compared with model group,both alcohol and water extracts from Clinacanthus nutans leaves significantly increased bone mineral density and maximum bone load of ovariectomized rats,which improved bone microstructure by increasing bone volume,bone volume fraction,bone surface,specific bone surface,trabecular thickness,connectivity density and reducing trabecular separation.The serum levels of bone alkaline phosphatase,tartrate-resistant phosphatase and c terminal telopeptide of collagen I were significantly decreased,and the levels of procollagen I n-terminal propeptide and bone gla protein were significantly increased.The results showed that the crude extract from Clinacanthus nutans leaves had significant effect on improving osteoporosis.Secondly,three active components,flavonoids,saponins and polysaccharides,were isolated and purified from the the alcohol and water extracts from Clinacanthus nutans leaves to explore theirs effects on osteoporosis in ovariectomized rats.The results showed that the content of flavonoids was 80 mg/100 mg,containing five flavonoids-C-glycosides and one flavonoids-O-glycosides.The content of saponins was 71 mg/100 mg,containing five triterpenoid saponins.The content of crude polysaccharide was 60 mg/100 mg,consisting of 8monosaccharides.The results showed that the active substances can reduce the level of serum interleukin 6,tumor necrosis factor-α and malondialdehyde,which further improve the levels of superoxide dismutase,catalase.Moreover the active substances could improve bone mineral density,bone maximum load,and restore bone trabecular partial mesh structure,which reverse the abnormal changes of serum bone transformation indicators in osteoporosis rats.These results suggested that flavonoids,saponins and polysaccharides of Clinacanthus nutans can inhibit inflammatory response and oxidative stress,and reduce the secretion of inflammatory factors and oxidative stress markers,and regulate bone metabolism,and improve osteoporosis in postmenopausal osteoporosis rats.Among them,saponins showed the most significant improvement effect on osteoporosis.Thirdly,the effects of Clinacanthus nutans saponins on proliferation,differentiation,mineralization and m RNA expression of osteogenic genes in MC3T3-E1 were investigated.The results showed that saponins had no toxic effect on MC3T3-E1 cells.After 24 h and 48 h treatment,saponins could significantly promote cell proliferation at the concentration of 500 and 1000 ?g/m L.After 3,6 and 9 days of treatment with different concentrations of saponins,the ability of osteoblasts to differentiate ALP could be significantly improved.After 14 days,saponin induced differentiation,the number of mineralized nodules was significantly higher than that of the control group.Clinacanthus nutans saponins could affect bone formation and bone resorption through OPG/RANKL and Wnt/β-catenin signaling pathways.Finally,the extraction process of total saponins from Clinacanthus nutans was optimized.The parameters of ultrasonic-assisted extraction of total saponins from Clinacanthus nutans leaves were determined by single factor and response surface methodology as follows: ethanol volume fraction 92%,solid-liquid ratio 1:44(g/m L),extraction time 0.6 h,extraction temperature 66℃.Under these conditions,the extraction rate of total saponins was the highest,which was 2.30%.In conclusion,the extracts of Clinacanthus nutans leaves have a potential effect on improving osteoporosis,among which saponins have the most significant effect on improving the function of osteoblasts.However,the effect of monomer components of saponins on osteoporosis and the mechanism of action remain to be further explored. |
