Metabolomics and Density Functional Theory Studies on the Antioxidant Capacity of Justicia gendarussa at Different Nitrogen Fertilizer Doses

Abstract

Justicia gendarussa, also known as gendarussa, is a plant that has the potential to be an antioxidant. J. gendarussa can grow wild as a bush, especially in forested settings and along river dams. These wild growth conditions will cause inconsistent secondary metabolite production and change biological activity, so it is necessary to cultivate J. gendarussa plants to maintain consistent secondary metabolite content. Plant cultivation treatments that can be carried out include different doses of nitrogen fertilizer. These metabolite changes can be observed using a metabolomics approach. Additionally, metabolomics can be used to identify active compounds. Then, to ensure the ability of the active compound, a computational density functional theory (DFT) chemistry study can be carried out. Therefore, this research aims to determine the optimal nitrogen fertilizer dose on J. gendarussa antioxidant capacity and metabolites using a metabolomics approach. Then, identify its antioxidant compounds in the J. gendarussa fraction with nitrogen fertilizer dose of the highest harvest weight using metabolomics and DFT. The research method begins with cultivating J. gendarussa plants at different doses of nitrogen fertilizer (0, 90, 180, 270 kg/ha), sample preparation, extraction, determination of extract antioxidant capacity, identification of extract metabolites, fractionation, determination of fraction antioxidant capacity, identification of fraction metabolites, DFT studies, and data analysis. The highest harvest weight with a mass of 11 g/plant was produced by plants given 270 kg/ha of nitrogen fertilizer. Antioxidant capacity was shown to be insignificantly different between treatments measured. Based on the metabolomics approach, variations in nitrogen fertilizer made metabolites at doses of 90 kg/ha and 180 kg/ha had higher relative concentrations of metabolites compared to doses of 270 kg/ha. So, 90 kg/ha are the optimal nitrogen fertilizer dose. The ethyl acetate and n-butanol fractions from the nitrogen fertilizer dose with the highest harvest weight had the highest antioxidant capacity. Principal component analysis (PCA) and heatmap succeeded in differentiating metabolites based on polarity. Orthogonal partial least square-discriminant analysis (OPLS-DA) succeeded in identifying six active compounds and 14 unknown compounds that had the potential to be isolated in the ethyl acetate fraction. DFT studies proves that 6-{[(Benzyloxy)carbonyl]amino}-6-deoxy-1,2-O-isopropylidene-alpha-D-glucofuranose and gendarusin A has a smaller gap energy than trolox standard, indicating its antioxidant activity is higher than trolox. Additional tests in vitro or in vivo research on the isolation results need to be carried out to prove the research results further.