Bioassay directed characterization of selected indigenous medicinal plants for their anti-HCV potential

Abstract

Hepatitis C is the most common chronic blood born infection affecting approximately 3 % population of the world and about 6 % population of the Pakistan. About one– fifth of the individuals with this infection results in the development of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. The present treatment standard is pegylated INF-a along with ribavirin, direct-acting antivirals (DAAs) like telaprevir and boceprevir for definite period according to viral genotype. The main goal of therapy is to achieve a stable virological response along with eradication of HCV infection and cure of the fundamental HCV induced liver disease. Unfortunately, only less than 50 % of HCV patients get benefit to some extent from this therapy due to side effects, resistance and high cost. Hence, there is a need to develop anti-HCV agents, both from herbal sources and synthetic chemicals which are non toxic, more efficacious and cost-effective. Soon-Valley of Pakistan is bestowed with a unique biodiversity, comprising of different climatic zones and wide range of plant species especially that have hepatoprotective effect. No doubt phyto-constituents having the remarkable potential inhibit the replication cycle of various types of DNA or RNA viruses. The present work is intended to explore plants with anti-HCV potential, leading to natural chemical entities as lead compounds. In current study, methanolic extracts of shade air dried selected parts of fifteen medicinal plants of Soon Valley were screened against HCV NS3 protease (genotype 3a) and as well as whole virus. The cellular toxicity effects of organic extracts on the viability of Huh-7 were studied through Trypan blue exclusion method and MTT assay. In serum inhibition assay, liver cells were infected with high titre of HCV positive serum of genotype 3a for screening of antiviral plants against whole virus. In in vitro protease inhibition assay, Huh-7 cells were transfected with HCV NS3 protease by introducing mammalian expression construct PCR3.1/FLAGtag/HCV NS3 in the presence and absence of plants extracts. Only the methanolic extract of Caralluma tuberculata (CTS) and Portulaca oleracea L. (POL) exhibited 57 % and 70 % inhibition. Four fractions of each CTS and POL extract were obtained through bioassay-guided extraction. Subsequent inhibition of all organic extract fractions against NS3 serine protease were checked to track the specific target inside the genome of the virus and in this case only ethyl aceatate and methanolic of CTS extracts inhibit the 69 % & 53 % and the POL extracts inhibit 80 % & 85 % expression of HCV NS3 protease respectively while keeping GAPDH expression constant. The results showed that the POL and CTS methanolic crude and ethyl acetate extract specifically abridged the HCV NS3 protease expression in a dose-dependent fashion. The plant organic extract was screened for the presence of phytochemical through standard procedures. Phytochemical analysis of selected fractions confirmed the presence of alkaloids, coumarins, flavonoids, glycosides, saponins, terpenoids, and tannins etc. In addition, the active fractions of both plant extracts were also checked for their some other biological activities like antioxidant potential, in vitro anti thrombotic and antibacterial activities. Antioxidant potential of CTSM, CTSE, POLM and POLE was determined in term of total phenol, flavonoids, tannin, carotenoid and free radical scavenging potential by following the different reported procedure. Free radicals scavenging activities of extracts were determined by DPPH, ABTS and FRAP assays. Among extracts highest antiradical values against DPPH were found to be 81.747 by CTSM while lowest value (36.124) revealed by POLE. Identification and quantification of phenolic acids in methanolic extracts of CTS and POL were done by HPLC with UV/DAD that confirm the presence of Sinapic acid, m-Coumaric acid, Gallic acid and Quercetin in CTSM along with Gallic acid and Quercetin in POLM. In case of in vitro anti thrombotic potential, highest activity among extract fractions was shown by CTSM and minimum by POLE while the overall order of antithrombotic potential was CTSM > CTSE > POLM >POLE. The antibacterial activity of methnolic extracts of CTS and POL against Gram positive (Bacillus subtilis, Styphylococcus aureus and Styphylococcus epidermidis) and Gram negative (Pseudomonas aerugenosa, Escherichia coli and Salmonella typhi) strains of bacteria was determined by disc diffusion method. Percent inhibition with respect to positive control (Ciproflaxacin) indicated that both CTSM and POLM exhibited more than 60 % inhibition against Styphylococcus epidermidis. Furthermore to identify the active ingredients, CTSE extract was also fractioned by column chromatography. Five compounds CT-EtOAc-01 i.e. Methyl(12-O-benzoylandrostan-3-O-(1→4)-β-D-cymaropyranosyl-(1→4)-β-Dcymaropyranosyl-(1→4)-β- D-cymaropyranosyl) hexanoate, CT-EtOAc-02 i.e. Methyl (12-O-benzoyl-androstan- 3-O-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl--(1→4)-β-Dcymaropyranosyl) hexanoate, CT-EtOAc-03 i.e. Methyl (23-O-benzoyl-androst-6-en- 17-O-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl--(1→4)-β-Dcymaropyranosyl)hexanoate, CT-EtOAc-04 i.e. Phenyl (12-O-benzoyl-gonan-6-en- 5’’-O-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl)oxalate and CTEtOAc-05 i.e. Trans-p-ferulylalcohol-4-O-(6-(2-methyl-3-hydroxypropionyl) glucopyranoside were isolated, purified and characterized by spectral data (UV-VIS, FTIR, EI-MS and NMR). The results obtained in this study suggested that methanolic and ethyl acetate extracts of CTS and POL possess remarkable antiviral, antioxidant, antithrombotic, and antibacterial effects which might be due to the charisma of their bioactive compounds like glycosides, flavonoids, tannins, saponins and alkaloids etc. Further studies in vitro, in vivo and in silico assays are mandatory to decipher the thorough aspects of these phytocompounds. The novel antiviral activity of compounds from CTS presents an attractive lead for natural chemical entities meant for the development of prospective anti-HCV agents. Hence, POL and CTS extracts and its potential constituents alone or plus with interferon could offer a future option to treat chronic HCV.