Screening of Selected Medicinal Plants from Pakistan for Phytochemicals, Antioxidants, Antimicrobial, Antiviral and Toxicological Activities

Abstract

Medicinal plants are being used extensively by man as safe alternative substitute for a number of synthetic medications to treat various ailments. In recent years, the interest in the use of plant derived natural compounds for developing antimicrobial and anticancer drugs has gained special attention. Plants may give new remedial arrangement as extracts or compounds which might be dynamic agents against pathogens. Therefore, demand for medicines from natural origin is increasing as they are generally safer, cost effective, nontoxic and less harmful than synthetic medications. Keeping in view the importance of medicinal plants, four indigenous medicinal plants were selected to evaluate the biological roles of their crude extracts. These include Ajuga bracteosa, Rumex dentatus, Foeniculum vulgare and Commelina benghalensis. These plants were used to prepare extracts with a range of solvents and then subjected to phytochemical analysis and biological evaluation including antioxidant, antibacterial, antifungal, antiviral and toxicological potential. Total phenolic and total flavonoid contents were estimated by Folin Ciocalteu and aluminium chloride methods respectively. Antioxidant potential was carried out by three commonly used methods such as reducing power assay, 1,1-diphenyl-2-picrylhydrazine (DPPH) radical scavenging activity and phosphomolybdenum method. Antibacterial activity of crude extract of selected plants was determined against six multi drug resistant strains and two sensitive reference bacterial strains through agar disc diffusion method. Antifungal activity was investigated against six pathogenic fungal strains by agar disc diffusion assay. All the selected plant extracts were also investigated for their cytotoxicity against hepatic cancer Hep2, prostate cancer DU145, breast cancer MDA-MB-231 and a normal breast cell line MCF-10A. The most active extracts were subjected to flow cytometry to analyze the changes in cell cycle and apoptotic effect. Cells were also studied for their wound healing and invasive potential as well as for Western blotting of apoptotic genes and nuclear factor kappaB (NFκB) pathway. Antiviral activity of four selected medicinal plants was examined against dengue virus serotype 2 (DENV 2) by plaque reduction neutralization assay using two DENV 2 doses (45 and 90 plaque forming units or PFU). Maximum quantity of extractable phenolic and flavonoid compounds was obtained in ethanol and methanol extracts while yield with n-hexane, chloroform and benzene was comparatively small. Their values for total phenolic contents ranged from ethanol extract of A. bracteosa (45.32 µg GAE/mg) to n-hexane extract of R. dentatus (9.29 µg GAE/mg) while highest (19.86 µg QE/mg) flavonoid contents were observed in ethanolic extract of C. benghalensis. Maximum antioxidant potential in terms of DPPH radical scavenging activity was measured for methanol extract of A. bracteosa (91.63 %, IC50 = 12.32 𝜇g/mL) however, maximum antioxidant capacity and reducing power were recorded for methanol extract of R. dentatus (23.47 µg AAE/mg) and ethanol extract of C. benghalensis (41.29 µg AAE/mg) respectively. Plants extract with higher amount of polyphenols were found with significant antioxidant potential and also a positive correlation was established among polyphenols and free radical scavengers. All the extracts showed varying degree of inhibitory action to bacterial and fungal strains. Among all the plant extracts, ethanol extracts of R. dentatus and F. vulgare proved highest antibacterial activity against Klebsiella pneumoniae 25 ± 1.42 mm (MIC: 25 𝜇g/mL) and 25 ± 1.89 mm (MIC: 25 𝜇g/mL) respectively. The highest antifungal effect was observed in case of ethanol extract of F. vulgare against Aspergillus flavus 19 ± 1.25 mm (MIC 25 𝜇g/mL) followed by methanol extract of C. benghalensis against Mucor species 18 ± 1.66 mm (MIC: 50 𝜇g/mL) and methanol extract of A. bracteosa for A. flavus 18 ± 1.45 mm (MIC: 50 𝜇g/mL). R. dentatus demonstrated highest cytotoxicity by its methanol and chloroform extracts against MDA-MB-231 cell line among all the tested cell lines with lowest IC50. So, methanol (RDM) and chloroform (RDC) extracts of R. dentatus and MDA-MB-231 cell line was selected for further analysis. The inhibitory effect in breast cancer cells was found to be linked with arrest of cell cycle (G0/G1 phase) along with induction of apoptosis by higher level of Annexin V positive cells and accumulation in sub G1 population. Moreover, both extracts (RDM and RDC) inhibited the proliferation and induced apoptosis by repressing the activation of NFκB and its subsequent transcripts (Bcl-xl, Bcl-2, cyclin D1, survivin and XIAP) in studied malignant cells. Apoptosis was also confirmed in breast cancer cells as suggested by caspase 3 detection. It was observed that RDM and RDC abrogate IκBa phosphorylation and constitutive NFκB activation in malignant cells. Moreover, it has been investigated that application of R. dentatus extracts in MDA-MB231 cells can reduce the invasion and migration ability of these cells. The study showed that all the studied plants are capable of inhibiting dengue virus by exhibiting prophylactic effect. Moreover, no plant was effective in chemotherapeutic assay. The methanol extract of R. dentatus demonstrated the highest antiviral efficacy by inhibiting DENV 2 replication, with IC50 of 0.154 μg/mL and 0.234 μg/mL, when added before infection with 45 and 90 PFU of virus, respectively. Present findings conclude that selected plants have antimicrobial, antioxidant, antiviral and cytotoxic potential and in the future, a complete investigation on plants for isolation, purification and characterization of active compounds should be carried out which are responsible for these activities.