IN VITRO ANTIOXIDANT CAPACITY OF THE FERN, DRYNARIA QUERCIFOLIA (L) SM., RHIZOME EXTRACT

Objective: The study was aimed to evaluate polyphenolic composition and antioxidant properties of methanol extract of rhizome of Drynaria quercifolia (L.) Sm. Methods: In this study, assessment of total phenolic and flavonoid contents were performed using Foiln-Ciocalteu and Aluminium chloride methods respectively. Antioxidant capacity of crude methanolic extract of rhizome of D. quercifolia were examined using established 1,1 – diphenyl – 2-picrylhydrazyl stable free radical (DPPH∙) assay, hydroxyl ion radicals (˙OH), nitric oxide (NO), hydrogen peroxide (H2O2)and 2, 2'-Azinobis (3-ethylbenzothiazoline sulphonic acid) ABTS•+ scavenging assays.  Results: The extract yielded total phenolic content (TP) of 240 ± 0.01 mg gallic acid equivalents (GAE)/100g of fresh mass (FM) and total flavonoid content (TF) of 150 ± 0.02 mg quercetin equivalents (QE)/100g of fresh mass (FM). The extract of D. quercifolia rhizome exhibited remarkable scavenging capacity towards DPPH∙ (EC50 18.54 ±0.70 µg mL-1), ˙OH (EC50 37.60±0.41 µg mL-1) NO (EC50 42.40 µg mL-1), H2O2 (EC50 32.80±1.89 µg mL-1) and ABTS•+ (EC50 29.80 ±0.70 µg mL-1). The antioxidant capacities of the extract were comparable and stronger than that of the antioxidant standard, butyl hydroxy toluene (BHT). The findings were also comparable with antioxidant properties of other medicinal ferns which are discussed in the study. Conclusion: Significant and positive correlations (R2 0.999-0.862) were observed between polyphenolic contents and the antioxidant capacities, indicating that the phenolics were major contributors of the antioxidant property. Thus, the methanol extract from the rhizome of D. quercifolia indicated the existence of strong antioxidants and it can be used as tonic

Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation

Hypoxia has diverse stimulatory effects on human adipose-derived stem cells (ASCs). In the present study, we investigated whether hypoxic culture conditions (2% O2) suppress spontaneous mineralization and osteogenic differentiation of ASCs. We also investigated signaling pathways and molecular mechanisms involved in this process. We found that hypoxia suppressed spontaneous mineralization and osteogenic differentiation of ASCs, and up-regulated mRNA and protein expression of Insulin-like growth factor binding proteins (IGFBPs) in ASCs. Although treatment with recombinant IGFBPs did not affect osteogenic differentiation of ASCs, siRNA-mediated inhibition of IGFBP3 attenuated hypoxia-suppressed osteogenic differentiation of ASCs. In contrast, overexpression of IGFBP3 via lentiviral vectors inhibited ASC osteogenic differentiation. These results indicate that hypoxia suppresses spontaneous mineralization and osteogenic differentiation of ASCs via intracellular IGFBP3 up-regulation. We determined that reactive oxygen species (ROS) generation followed by activation of the MAPK and PI3K/Akt pathways play pivotal roles in IGFBP3 expression under hypoxia. For example, ROS scavengers and inhibitors for MAPK and PI3K/Akt pathways attenuated the hypoxia-induced IGFBP3 expression. Inhibition of Elk1 and NF-κB through siRNA transfection also led to down-regulation of IGFBP3 mRNA expression. We next addressed the proliferative potential of ASCs with overexpressed IGFBP3, but IGFBP3 overexpression reduced the proliferation of ASCs. In addition, hypoxia reduced the osteogenic differentiation of bone marrow-derived clonal mesenchymal stem cells. Collectively, our results indicate that hypoxia suppresses the osteogenic differentiation of mesenchymal stem cells via IGFBP3 up-regulation