Phytochemical Analysis, In Vitro Free Radical Scavenging, and LDL Protective Effects of Different Solvent Fractions of Calotropis procera (R.) Br. Root Bark Extract

Abstract

In the present study, the methanolic extract of Calotropis procera root bark was subjected to solvent-solvent partitioning using n-hexane fraction (HF), dichloromethane fraction (DMF), ethyl acetate fraction (EAF), and methanol fraction (MF). The resultant fractions were tested for antioxidant activity using in vitro radical scavenging assay. Preliminary phytochemical investigation revealed the presence of varying proportions of secondary metabolites in solvent fractions such as glycosides, flavonoids, triterpenoids, sterols, and polyphenolic compounds. The total phenolic content of EAF was 25.7 ± 3.12 mg TAE/g followed by 19.05 ± 3.29 mg TAE/g in DMF. The total flavonoid content was 13.69 ± 1.74 mg QUE/g in DMF and 11.4 ± 1.88 mg QUE/g in EAF. The EAF showed significant radical-scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH, IC50  = 369.87 μg/mL), nitric oxide (IC50  = 317.46 μg/mL), hydrogen peroxide (H2O2 , IC50  = 396.85 μg/mL), and hydroxyl radicals (IC50  = 195.39 μg/mL). DMF was most effective in scavenging superoxide radicals (IC50 = 679.60 μg/mL), while greater metal chelating activity was exhibited by MF (IC50 = 614.73 μg/mL). Moreover, the total antioxidant activity for EAF was found to be 94.14 ± 9.114 mg AAE followed by DMF (68.10 ± 8.78 mg AAE). EAF also significantly reduced the formation of thiobarbituric acid reactive substances in a dose-dependent manner in CuSO4. The observed antioxidant effect might be attributed to the presence of secondary metabolites. Subsequent GC-MS analysis of EAF confirmed the presence of lupeol, α-amyrin, β-amyrin, and ursolic acid. The current investigation reveals that the high polyphenolic and antioxidant pentacyclic triterpenes in EAF of C. procera root bark methanol extract correlates with its good antioxidant activity and can provide protection against free radicals-induced damage in a variety of chronic health conditions.