Biological activity of HPLC-characterized ethanol extract from the aerial parts of Haplophyllum tuberculatum

Abstract

Context: The search for new sources of natural antioxidants from plant material may have beneficial therapeutic potential for those diseases associated with oxidative stress. The medicinal plant Haplophyllum tuberculatum (Forsskal) A. Juss. (Rutaceae) contains phenolic compounds as main phytochemicals; however, there are no reports on its antioxidant properties.

Objective: To evaluate antioxidant and cytoprotective potential of ethanol extract of Haplophyllum tuberculatum aerial parts.

Materials and methods: Total phenol content was determined using Folin–Ciocalteu reagent; antiradical activity was measured using ORAC assay and the analysis of the major polyphenols was carried out using a HPLC-MS method. The antioxidant and cytoprotective effect were also investigated by the MTT assay and DCFH–DA method. The human astrocytoma U373-MG cell line was pretreated with ethanol extract (from 0.025 to 250 µg/mL) for 24 h, prior to 1 mM H₂O₂ exposure (30 min).

Results and conclusion: Total phenol content was 46.2 mg gallic acid/g sample and ORAC value was 1.283 µmol TE/mg sample. Chemical constituents were methoxyflavones, flavonols (mainly quercetin derivatives), cinnamic acids and benzoic acids. In cell system model of oxidative stress, pretreatments with ethanol extract at the concentrations of 2.5, 0.25 and 0.025 µg/mL significantly attenuated H₂O₂-induced loss in viability by 13.5, 17 and 20.5%, respectively. Furthermore, these ethanol extract concentrations markedly inhibited intracellular ROS production with IC₅₀ 0.026 µg/mL. These findings demonstrate the beneficial properties of ethanol extract of Haplophyllum tuberculatum aerial parts, rich in phenolic compounds, as antioxidant and radical scavenger ameliorating ROS-related processes and diseases such as several neurodegenerative disorders.

• Antioxidant activity
• astrocytes
• free radicals
• hydrogen peroxide
• polyphenols

Acknowledgements

The authors thank Drs I. Estrella and T. Hernández from Instituto de Ciencia y Tecnología de Alimentos y Nutrición (CSIC), Madrid, Spain for technical assistance with HPLC analysis.