Biochemical and Antimicrobial Properties of Secondary Metabolites of Withania somnifera Dun

Abstract

Withania somnifera is an important medicinal plant traditionally used in the treatment of many diseases. Plant derived natural products such as phenolic compounds, flavonoids, anthocyanin, ascorbic acids, terpenoids and steroids etc. have received considerable attention in recent years due to their diverse pharmacological properties including antioxidative, antidiabetic, anticarcinogenic, antimicrobial, antiallergic, antimutagenic and anti-inflammatory activities. The present study was carried out to characterize the secondary metabolites, especially, phenolic acids, flavonoids and antioxidant as well as antimicrobial properties in methanolic extracts of W somnifera fruits (WSFEt), roots (WSREt) and leaves (WSLEt). WSFEt, WSREt and WSLEt were prepared by with 80% aqueous methanol and tested for the presence of phytochemicals using standard qualitative procedures. Total polyphenols, flavonoids, anthocyanin and ascorbic acids were determined spectrophotometrically. DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging activities, ferric reducing antioxidant power (FRAP) assay, ferrous chelating activity, reducing power assay and Inhibition of 13-carotene bleaching, were also determined by spectrophotometric methods, DNA damage protective activity was determined by agarose gel electrophoresis and finally phenolic acid profiles were determined by HPLC methods. The antibacterial activity of WSREt, WSFEt and WSLEt was determined by agar well diffusion method. Preliminary phytochemical analysis of WSFEt, WSREt and WSLEt revealed the presence of glycosides, alkaloids, phytosterols, fixed oils, phenolic compounds and flavonoids. High concentrations of both phenolics and flavonoids were detected in all parts of the plant with the former ranged between 17.80 ± 5.80 and 32.58 ± 3.16 mg/g (dry weight) and the latter ranged between 15.49 ± 1.02 and 31.58 ± 5.07 mg/g. The ascorbic acid content ranging between 62.60 mg/1 00g and 20.60 mg/1 00g. The total anthocyanin contents of WSLEt, WSFEt and WSREt were 12.5 ± 1.04 mg/1 00g, 5.66 ± 0.52 mg/100g and 2.86 ± 1.44 mg/100g respectively. All of the three different plant parts showed strong DPPH radical scavenging activities (59.16 ± 120% to 91.84 ± 0.38%) and its IC50 values ranged between 101.73 µg/ml and 801.93 µg/ml. FRAP assay of WSLEt, WSFEt and WSREt showed strong antioxidant properties and the highest value (1643.04 µM Fe/kg) was found in leaves while the lowest value (1130.18 µM Fe/kg) was found in roots. Ferrous chelating activity (IC50) of the WSLEt, WSREt, WSFEt ranged between 0.22 ± 0.04 mg/ml and 0.65 ± 0.02 mg/ml. Reducing power assay among the three extracts, WSLEt showed higher absorbance compared to WSREt and WSFEt. Lipid peroxidation of WSLEt, WSREt and WSFEt exhibited 79.67%, 69.87%, and 72.11 % peroxidation of linoleic acid emulsion at the concentration 60 µg/ml, respectively. DNA damage protective activity was also found at different concentration (2.5, 5, 10, 20 µg/ml) of WSLEt, WSREt and WSFEt. Eight polyphenols (gallic, syringic, benzoic, p-coumaric and vanillic acids as well as catechin, kaempferol and naringenin) were identified by HPLC as well. Among all the polyphenols, catechin was detected in the highest concentration (13.01 ± 8.93 to 30.61 ± 11.41 mg/g). Sensitivity test of WSREt, WSFEt and WSLEt showed strong antibacterial activity against five human pathogenic bacteria E. coli, Salmonella, Citrobacter, Pseudomonas and Klebsiella. WSLEt showed the highest zone of growth inhibition against S. typhi. The antifungal screening showed that methanolic extract of W somnifera roots, fruits and leaves was active against Fusarium oxysporum and increasing concentration of the extract showed higher inhibitory effect of the fungus and W somnifera roots extract have showed the highest activity compared to fruits and leaves. The results indicate that W somnifera is a plant with strong therapeutic properties thus further supporting its traditional claims. All major parts of W somnifera such as the roots, fruits and leaves provide potential benefits for human health because of its high content of secondary metabolites especially phenolic compounds, flavonoids, ascorbic acids and anthocyanin as well as antioxidant and antimicrobial activity. W. somnifera leaves contain the highest amounts of polyphenols specially catechin which has promising medicinal and pharmacological value.