Physical and Chemical methods of extraction of Bioactive Molecules from Lepidium sativum Linn. and Antioxidant Activity-based screening and selection of extracts-Probable Phytochemical, Chromatography and mass spectroscopy analysis-based correlates

Abstract

Identification and isolation of active principles from Lepidium sativum L. garden cress seeds and their chemical/biological characterization would aid in drug development (templates for synthesizing derivatives or as a value-added product). Initially, this study dealt with the phytochemical, qualitative and quantitative analysis for phenolic and flavonoid compounds. Subsequently, different physical and chemical extraction techniques -Simple Crude extraction (CRU), Ultra Sonication-Assisted Extraction (UAE), Microwave-Assisted Extraction (MAE) and classical Soxhlet extraction were adopted to select the best Lepidium sativum L. seed extract based on their activity in certain classical, in vitro antioxidant assay systems. The selected best extract was further analyzed for characterization using UPLC, GC-MS. CRU MeOH extracts showed a higher Total phenolic content of 4464.1±349.7mg GAE/100g. The UAE method showed a higher total flavonoid content of 1520.6±182.2mg QUE/100g. CRU-MeOH showed good antioxidant activity based on the free radical scavenging DPPH assay (IC50: 50.61µg/mL). A 100µg/mL concentration of CRU-MeOH was found to be 217.82±12.82 FRAP value. At 400 µg/mL, the NO scavenging assay was reported to be 62.11±4.84%. The present study indicates that extracts from the polar solvents shows better antioxidant scavenging potential than that of the non-polar extracts. Based on the relatively superior performance of the CRU-MeOH extract, UPLC-PDA data was generated. The validity of our chromatography conditions was shown by the presence of quercetin at a retention time 8.555 (validated by our internal standard), apart from the presence of this important bioactive molecule in our extract. As another strand of evidence for the presence of important bioactive molecules, p-coumaric acid, and ferulic acid were also present in our extracts, following correlation of their UPLC-PDA data with published findings. Also, GC-MS analysis of the CRU MeOH extract showed that 24 compounds (8 major and 16 minor peaks). One of the compounds detected (cis-vaccenic acid) is in consonance with published data. The presence of bioactive components, with known antioxidant and cell death potential, validates our experimental flow for the development and/or refinement of crude extract-based drugs. However, the variability perforce warrants an inter-laboratory harmonization of protocols for making meaningful comparisons.