This study is the first to report and compare the antioxidant and antibacterial properties of the different parts of W. somnifera, namely the leaves, roots and fruits. Our study is also the first to report on the ascorbic acid levels of WSFEt and WSLEt as well as the anthocyanin concentrations present in W. somnifera. We found that W. somnifera leaves have the highest antioxidant and antimicrobial activities as well as the highest ascorbic acid and anthocyanin contents. These observations may be related to the high phenolic and flavonoid contents reported in our previous study
The highest concentration of ascorbic acid was found in WSLEt, whereas WSREt had the lowest ascorbic acid content. Hussain et al.,
 reported a higher vitamin C content (51.50 mg/ 100 g) in WSREt. In another study
, the ascorbic acid contents of the leaves and roots of W. somnifera were compared, and the roots were found to possess a slightly higher ascorbic acid content. This variability may be due to differences in the source of the W. somnifera plant used. Anthocyanins are flavonoids
 and are among the important phenolic substances in W. somnifera. Flavonoids are polyphenolic compounds with known properties that include free radical scavenging, inhibition of hydrolytic and oxidative enzymes and anti-inflammatory action
. The mechanisms of action of flavonoids are through scavenging or chelating processes
. It has been reported that the anthocyanins present in W. somnifera are chemically-similar to the ones present in black raspberry (Rubus occidentalis)
. As with ascorbic acid, the content of total anthocyanins in WSLEt, WSREt and WSFEt varies in different parts of the plant and is highest in the leaves. However, this level is lower than the highest concentration of anthocyanins found in tomato, which is 2.83 ± 0.46 mg/g
Free radical DPPH scavenging ability is extensively used to indicate the antioxidant potential of naturally derived foods and plants. We found that W. somnifera exhibited good DPPH free radical scavenging abilities. WSLEt had the lowest IC50. IC50 values between 206.77 and 224.96 μg/ml
 have been reported in previous studies involving only W. somnifera leaves. These researchers indicated that the IC50 values for plant extracts harvested from polluted sites are higher when compared with those harvested from non-polluted sites.
A relatively higher FRAP value, as was determined for WSLEt, indicated a greater reduction of ferric ions to ferrous ions and higher antioxidant properties in the leaves when compared with the other plant parts (Table
Metal chelating capacity is important because it reduces the concentration of transition metals that catalyze lipid peroxidation
. Ferrous chelation tests were performed, and the results showed that although WSREt, WSFEt and WSLEt did not display chelation activities as high as that of the EDTA standard (Table
2), the decrease in the concentration-dependent color formation in the presence of the extracts indicated that they did exhibit some amount of iron-chelating activity.
However, W. somnifera extracts exhibited high inhibitory effects to lipid peroxidation of linoleic acid. The activities of antioxidants have been attributed to various mechanisms such as prevention of chain initiation, decomposition of peroxides, reducing capacity and radical scavenging
. High inhibitory effect of lipid peroxidation of the extracts could be due to the abundant presence of antioxidant active compounds. Therefore, these extracts may be effective agents in retarding Fe2+ catalyzed lipid oxidation.
These results indicate that the antioxidant capacity of each extract may be related to the concentrations of ascorbic acid, anthocyanin and polyphenols. The antioxidant activities of these compounds depends on their molecular structures, that is, on the availability of phenolic hydrogens which result in the formation of phenoxyl radicals due to hydrogen donation
For the antibacterial activity study, the 80% methanolic extract of all parts of W. somnifera displayed activity against five pathogenic Gram-negative bacteria, namely E. coli, S. typhi, C. freundii, P. aeruginosa and K. pneumoniae, to different magnitudes. W. somnifera leaves possessed the greatest antimicrobial effects. Phenolics, ascorbic acid and anthocyanins are associated with the antimicrobial efficiency of the plant because they cause hyperacidification at the plasma membrane interface of the pathogen, which potentially results in the disruption of the H+-ATPase required for ATP synthesis
The most susceptible organism was S. typhi, indicating that the W. somnifera extracts contain active compounds that can inhibit the proliferation and growth of S. typhi. which can cause diseases such as typhoid fever and foodborne illnesses. This finding may support the traditional uses of W. somnifera as a therapeutic agent for diarrhea, dyspepsia and gastrointestinal disorders
. The other tested bacteria also exhibited significant sensitivities against WSREt, WSFEt and WSLEt. These results demonstrate that the methanolic extracts contained the expected compounds for antibacterial activities against the five tested Gram-negative bacteria. W. somnifera may be exploited as a natural drug for the treatment of several infectious diseases initiated by these organisms. This finding is important in the quest for new antimicrobial agents because organisms with multidrug resistance are rapidly emerging.
Jain and Varshney
 reported on the antibacterial activity of the methanolic extracts of the whole W. somnifera plant against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans and Candida albicans, with zones of inhibition of 38, 36, 15, 38 and 32 mm, respectively. These results are similar to our reported zones of inhibition for WSLEt against two of the same organisms, Escherichia coli and Pseudomonas aeruginosa, at 28 ± 0.56 and 26 ± 1.08 mm, respectively. However, Jain and Varshney
 reported that aqueous extracts of W. somnifera had higher antimicrobial activities (a zone of inhibition between 33 and 50 mm) when compared with methanolic extracts. In this study, methanol was used to extract low molecular weight and moderately polar substances because of its wide range of solubility. However, Jain and Varshney
 did not report any MIC values for comparison with our own values.
Ascorbic acid, anthocyanin and polyphenols have been reported to exhibit antibacterial activities with distinguishing characteristics for their reactivities with proteins related to polyamide polymers
. The inhibition of microorganisms by these antioxidant compounds may be due to iron deprivation or hydrogen bonding with vital proteins such as microbial enzymes
. Antioxidant compounds such as ascorbic acid, anthocyanin and polyphenols are vulnerable to polymerization in air through oxidization reactions. Therefore, an important factor governing their toxicity is their polymerization size. It has been reported that the oxidized condensation of antioxidant compounds may result in the toxification of microorganisms
. These findings support the fact that ascorbic acid, anthocyanin and polyphenols may be responsible for the antimicrobial activities of WSREt, WSFEt and WSLEt.
The phytochemical constituents quantified in the present study such as ascorbic acid as well as anthocyanin and as also determined in our previous study i.e. the polyphenols and flavonoids
 are of significant medicinal importance and may act as antioxidant, antimicrobial and immunomodulatory agents. In the future, the above phytoconstituents could be used as a major tool for obtaining a quality control profile for a drug.