ASSESSMENT OF ANTIMICROBIAL, ANTIOXIDANT ACTIVITIES AND PHYTOCHEMICAL SCREENING OF LEPTADENIA PYROTECHNICA

Abstract

Leptadenia pyrotechnica (Forssk.) Decne (Asclepiadaceae), a medicinal plant is native to hot deserts of Pakistan. The present study was designed to assess antimicrobial and antioxidant activities and phytochemical screening of this plant. Eight solvents based extracts viz., hexane, chloroform, acetone, ethyl acetate, butanol, ethanol, methanol and water were prepared from the roots and aerial parts of the plant for phytochemical screening and antimicrobial activity while antioxidant activity of b Preliminary phytochemical screening involved qualitative and quantitative screening of four major groups of phytochemicals including alkaloids, flavonoids, saponins and tannins. Qualitative screening was carried out by simple biochemical tests that revealed the presence of all major groups of phytochemicals in both parts of the plant. Methanol was the most efficient solvent that extracted all the selected classes of phytochemicals. It was followed by ethanol, which also reflected a good extraction efficiency. The percentage of alkaloid contents was 3.267±0.643 and 3±0.6 in roots and aerial parts respectively (p>0.05). The total flavonoid content was 76.867±2.266 and 139.448±8.677 QE/100g of extract in roots and aerial parts respectively. The total saponin contents were 0.34±0.013% and 0.46±0.010% in roots and aerial parts respectively. The total tannin contents were 62.713±4.841 and154.961±5.853 mg of TAE/100g of extract in roots and aerial parts respectively. For the determination of antimicrobial activity, agar well diffusion method was employed utilizing eight solvent extracts against Staphyllococcus aureus and S. 18 epidermidis and two fungal strains viz. Aspergillus fumigatus and A. niger, which are the causative agents of various human infections. Antifungal activity was very weak while antibacterial activity was appreciably good. Both plant parts had significant differences in inhibiting bacterial growth (p<0.05). Root extracts were found more effective than the aerial parts extracts in checking bacterial growth. The root extracts inhibited S. epidermidis and S. aureus with the Zone of inhibition (ZI) that was 15+1.73 and 13+1.73mm respectively, followed by the aerial parts extract (ZI: 10±0.58 and 10±1.53mm respectively). The methanolic root extracts exhibited promising antibacterial activity (Acitivty Index: 0.1) that inhibited the growth of S. epidermidis at par with the standard antibiotic. With reference to solvent extracts, methanolic ones were the most effective in inhibiting bacterial growth resultantly minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was determined against S. aureus and S. epidermidis by using tube dilution method. The root extract exhibited pronounced effect on S. epidermidis with the MIC of 12.5mg/ml. On the other hand, S. aureus was also inhibited by root extract with the MIC of 25mg/ml. Likewise; MBC of root extracts was 20 mg/ml and 30 mg/ml against S. epidermidis and S. aureus respectively. The MIC of aerial parts extracts was 25mg/ml and 50mg/ml against S. aureus and S. epidermidis respectively. Likewise, MBC of aerial parts extracts was 25mg/ml and 12.5mg/ml against S. aureus and S. epidermidis respectively. Antioxidant activity of methanolic extracts of both plant parts was determined at ten different concentrations ranging from 10 μg/ml to 100 μg/ml through three 19 methods viz., 1) DPPH scavenging, 2) hydrogen peroxide scavenging and 3) reducing power assays. Both plant parts showed strong antioxidant capacity determined through all assays. There was significant difference in activity expressed by all selected concentrations amongst the three assays (p<0.05). Furthermore, the activity was found directly proportional to concentration. The antioxidant activity of this plant depicted by all assays was comparable with that of synthetic antioxidant agent i.e. Butylated Hydroxy Anisole (BHA). The results of bioactivity exhibited the efficiency of methanolic extracts. Bioactivity guided study of the extracts was carried out through three different techniques including 1HNMR Spectroscopy, HPLC and LC-MS. For this purpose, NMR and LC-MS based metabolomics analysis of all solvent extracts coupled with multivaritate statistical analysis including Principal Component Analysis (PCA) and Partial Least Square Discriminant Analysis (PLS-DA) was carried out. The analysis of NMR based spectral data confirmed the metabolic differences and similarities in different solvent extracts of both parts of L. pyrotechnica. On the other hand, analysis of LC-MS based chromatographic data predicted four components as potential antibacterial agents. The methanolic extracts were analyzed through HPLC and the fractions obtained through Reverse Phase HPLC were analyzed against bacterial pathogens, where none of the fractions exhibited activity, which reflected that more than one compounds might be acting synergistically in inhibiting bacterial growth. It can be concluded that extracts from both plant parts showed appreciable antibacterial activity as well as antioxidant activity along with range of 20 phytochemicals. The antibacterial and antioxidant activities of the plant validated scientifically the traditional use of this plant for treating various human diseases by the natives of desert habitats of Pakistan. The findings are stressing the need for further indepth analysis of extracts from the said plant. Such findings may lead to identification of potential compounds responsible for antimicrobial and/or antioxidant activities. In addition, in vivo assays may be conducted in future to assess the potential toxicity of the extracts that may ultimately lead to drug development.