Phenolics compounds from the roots of Anthospermum emirnense Baker (Rubiaceae)

Background: Anthospermum emirnense Baker (Rubiaceae), an indigenous and endemic plant of Madagascar, is widely used in traditional Malagasy medicine. The juice extracted from its crushed roots is traditionally applied to treat toothaches and skin infections. The aim of this study was to evaluate its antioxidant activity, quantify its total phenolic content, and isolate and identify its phytochemical constituents.

Methods: The roots of the plant were collected, shade-dried, and pulverized into a fine powder. Sequential extraction was performed by maceration using solvents of increasing polarity: hexane, ethyl acetate, and methanol. Preliminary phytochemical screening of the crude ethanol extract was conducted using standard qualitative methods. The total polyphenol content of the extracts was determined spectrophotometrically using the Folin–Ciocalteu method. Qualitative and quantitative antioxidant tests were performed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical. Acute toxicity tests were also carried out.

All experimental results were statistically analyzed using analysis of variance (ANOVA) followed by Tukey’s post hoc test. Chromatographic separation of the extracts was performed, and compounds isolated from the ethyl acetate and methanol extracts were identified using nuclear magnetic resonance (NMR) spectroscopy.

Results: Biological assays demonstrated low antioxidant activity for both the ethyl acetate (AcOEt) and methanol extracts, with 50% inhibitory concentrations (IC₅₀) of free radicals measured at 155.02 ± 2.6 µg/mL and 226.53 ± 0.1 µg/mL, respectively. The total phenolic content of each extract was determined to be 168 mg gallic acid equivalents (GAE)/g of dry extract (DE) for the crude ethanol extract, 34 mg GAE/g DE for the ethyl acetate extract, and 38 mg GAE/g DE for the methanol extract. Fractionation of the ethyl acetate and methanol extracts led to the isolation of five known compounds: one anthraquinone, 6-hydroxyrubiadin (1); one naphthoquinone, dehydro-α-lapachone (2); one coumarin, 6-hydroxycoumarin (3); and two iridoids, 8-acetyl-1-epishanzigenin methyl ester (4) and 8-acetylshanzigenin methyl ester (5).

Conclusion: These findings demonstrate the presence of phenolic compounds in the crude ethanolic, ethyl acetate, and methanolic extracts of Anthospermum emirnense roots. The extracts exhibit moderate antioxidant activity and show signs of acute toxicity. These results highlight the potential of this plant for use in phytomedicine. However, the relatively low antioxidant activity suggests a low polyphenol content.