Molecular studies of dioscorea birmanica Prain & Burkill extract and its subfractions on protective effects against oxidative stress and inflammation in cellular models

The rhizomes of Dioscorea birmanica Prain & Burkill (DB) have long been used as the main ingredient in Thai remedies for treatments of chronic diseases whose molecular pathogenesis is linked to long-term oxidative stress and inflammation. Nevertheless, its protective effects against oxidative stress and inflammation have never been studied. Therefore, this research aimed to investigate the protective effects of the ethanolic extract of DB rhizomes (DBE) against oxidative stress-induced normal liver cells and the anti-inflammatory activities of the most potent fraction of DBE against inflammation-induced THP-1 macrophages. DB rhizomes were extracted with 95% ethanol to obtain DBE, which was further assessed for antioxidant actions using multiple chemical assays. Moreover, protective effects of DBE were studied in normal liver BNL CL.2 cells against sodium nitroprusside (SNP)-induced oxidative stress by measuring cell viability, biomolecule damage, and antioxidant enzyme activities. Molecular mechanisms underlying the protective effects of DBE were verified by measuring intracellular free radical levels in BNL CL.2 cells. Total contents of phenolics, flavonoids, and saponins were measured by colorimetric assays, and some of them were structurally identified by LC-ESI-QTOF-MS/MS. As for anti-inflammatory studies in THP-1 macrophages, 4 fractions were obtained from DBE by sequential partitioning in hexane:chloroform, chloroform, chloroform:methanol, and methanol systems using vacuum liquid chromatography. Total contents of phenolics, flavonoids, and saponins of 4 fractions were measured by colorimetric assays. The fraction containing the highest content of these compounds was assumed to exert high anti-inflammatory activities and further isolated to obtain pure compounds using column chromatography. After that, all fractions were evaluated for the most potent fraction against lipopolysaccharide (LPS)-induced inflammation through measurements of cell viability using the resazurin assay and of inhibitory activity against COX-2 protein expression using the western blot analysis. Further, the most potent fraction was investigated for the anti-inflammatory activities against TNF-α and IL-6 mRNA expression by the real-time PCR. In addition, phytochemical compounds were structurally identified by LC-ESI-QTOF-MS/MS. The results revealed that DBE possessed effective in vitro scavenging activities against synthetic and natural radicals and in metal ion chelating activity. For protective studies, DBE significantly increased the cell viability, reduced biomolecule damage on lipids, proteins, and DNA, and restored the antioxidant enzyme activities against SNP-induced oxidative stress compared to DBE-untreated cells. In parallel with its cytoprotection, DBE effectively reduced high levels of intracellular free radicals, thereby revealing that its protective mechanism was via the reduction of oxidative stress, which was attributed to its potent antioxidant activities, subsequently ameliorating oxidative damage. For phytochemical compounds, DBE had high levels of phenolics, flavonoids, and saponins, and 14 of which were potential antioxidants with elucidated structures. As for studies on anti-inflammatory activities, among 4 fractions, the chloroform fraction contained the highest content of phenolics, flavonoids, and saponins, and pure compounds, namely diosgenin and 5-(2-(3, 5-dimethoxyphenyl)ethyl)-2-methoxyphenol, were isolated from this fraction and identified. In addition, studies on anti-inflammatory activities in LPS-induced THP-1 macrophages revealed that the hexane:chloroform fraction was the most potent in increasing cell viability and suppressing COX-2 expression. Additionally, this fraction significantly inhibited expression levels of TNF-α and IL-6 mRNA. For phytochemical compounds, the hexane-chloroform fraction contained high levels of saponins, some of which were structurally elucidated. DBE has the potential source to develop into herbal dietary supplements for protection against oxidative stress and inflammation.