Comparative metabolic profiling, isolation of alkylated phenols and antioxidant activity of roots of Plumbago species using GC-MS and NMR based metabolomics study

Abstract In this study, NMR and GC-MS based comparative metabolomic profiling of the roots of three different species namely, Plumbago indica, P. auriculata and P. zeylanica were investigated followed by multivariate statistical analyses and their antioxidant activity. Also, two alkylated phenols i.e., 2,6-di-tert-butyl phenol and 2,4-di-tert-butyl phenol not reported earlier from this taxon were isolated from P. indica. This metabolic study resulted in the identification of 25 and quantification of 18 metabolites. Principal component analysis showed the clear distinction among the three species. The antioxidant activity in the extracts was tested by free radical scavenging method. The three Plumbago species revealed interesting antioxidant potential, in particular, P. indica, which was rich in naphthoquinones, coumarins, alkylated phenols, sterols, triterpenes, fatty acids showed lowest IC50 value. The results highlighted the role of P. indica in the management of oxidative stress especially when they are utilized in the formation of fermented food products. Graphical Abstract


Introduction
Genus Plumbago belongs to the Plumbaginaceae family is native to India, Africa, Australia, Indonesia and Southeast Asia (Chopra et al., 1956). The root and chemical constituents of Plumbago indica Linn., P. auriculata Linn. and P. zeylanica Linn. are credited to own therapeutical properties according to the Indian traditional medicine system (Jain et al. 2014). These species have been reported for their nutritional and antioxidant potential which is associated with their richness in naphthoquinones, sterols, terpenes and fatty acids content (Kaewbumrung and Panichayupakaranant 2012). Metabolomics is a powerful approach which has gained widespread applications in order to understand the variations in primary and secondary metabolism in closely related species of plants and animals (Sayed et al. 2020).
Since no attention has been paid to Plumbago species variation associated with secondary metabolites, this study was constructed to compare the roots of Plumbago indica, P. auriculata and P. zeylanica using GC-MS and NMR based metabolomics approach. Also, first time isolation of two alkylated phenols from the roots of P. indica was conducted. Additionally, we investigated their in vitro antioxidant capacities so that nutraceuticals and food supplement industries get benefitted from such findings.

Multivariate statistical analysis
A principal component analysis (PCA) was applied on GC-MS and NMR obtained data in order to examine the intrinsic metabolites variation among P. indica, P. auriculata and P. zeylanica. According to PCA score plot ( Figures S3A-4A), the three species of Plumbago were clustered away from each other indicating different metabolic concentration in each species. 2,6-DTBP and 2,4-DTBP, fatty acids, sterols, triterpenes, indole derivatives, anthranilic acid, b-asarone, naphthoquinones and coumarins were most abundant and characteristic metabolites in P. indica, notably alkanes, alkenes and plumbagic acid which were significantly more abundant in P. auriculata and P. zeylanica ( Figures S3B-4B).

Antioxidant potential
To investigate the antioxidant potency of 2,6-DTBP and 2,4-DTBP, hexane and methanol extracts of P. indica, P. auriculata and P. zeylanica roots towards the most common reactive oxygen species (ROS), obtained extracts were subjected to in vitro antioxidant assay, namely DPPH radical scavenging activity. As shown in Figure S5, compound 2,6-DTBP showed a remarkable radical scavenging activity (IC 50 ¼ 42.26 lg/mL) against control ascorbic acid and BHT. Pertains to this, hexane and methanol root extracts of P. indica partially endowed this activity (IC 50 ¼ 124.11 and 82.41 lg/mL) in comparison to P. auriculata (162.74 and 91.63 lg/mL) and P. zeylanica (188.57 and 95.28 lg/mL) These results are highly correlated to high content of alkylated phenols, naphthoquinones, coumarins, fatty acids, triterpenes and sterols in P. indica roots.

Conclusions
Results reported here clearly highlighted that the naphthoquinones and other secondary metabolites variation based on GC-MS and NMR is able to discriminate the three species of Plumbago viz., P. indica, P. auriculata and P. zeylanica. The present investigation revealed the efficiency of metabolomics in chemotaxonomical characterization of closely associated plant species. P. indica was characterized by its richness in naphthoquinones, coumarins, alkylated phenols, triterpenes, fatty acids and sterols and showed a broad-spectrum in vitro antioxidant potential. P. indica are currently becoming endangered threatened species because of continuous use in herbal, pharmaceutical and nutraceutical industries. Such biological specimens should be cultivated so that the roots of P. indica can be utilized in the production of value-added fermented food products.