High-throughput multiparametric live-cell and immunofluorescence analyses demonstrate antifibrotic bioactivities of O. basilicum and A. membranaceus hairy roots on human hepatic stellate cells

Plants are a well-established source of compounds with biopharmaceutical activity, but the diversity and often low abundance of phytochemicals have hindered their medical application. Bridging this gap requires scalable growth conditions and sustainable extraction methodologies harnessed to robust and predictive assays of disease and cell toxicity. This study utilises plant hairy root biomass and constituent bioactives combined with high-throughput assays of fibrogenic progression using activated human hepatic stellate cells. Fibrosis is a build-up of connective tissue components like collagen type I and fibronectin and is one of the underlying drivers of liver cirrhosis, a complex and often fatal condition arising from alcohol abuse, poor diet, or viral infection. New treatments are required to reverse the predicted steady increase in the global burden of this disease. In this study, Astragalus membranaceus and Ocimum basilicum were transformed with Agrobacterium rhizogenes ( Rhizobium rhizogenes ) to provide reproducible and rapidly growing hairy root cultures. Titrations of methanol and pressed root extracts obtained from both species, altered selectively, the deposition of collagen type I and fibronectin, along variable effects on live readouts of stellate cells proliferation and death, distinguishing between Caspase-3/7 activation and membrane integrity maintenance. These results differ from the wider effects observed with the known anti-fibrotic compounds Nintedanib and Silymarin, which suggests that the extracts may prove to be a significant source of novel biopharmaceuticals useful in the treatment of liver cirrhosis. Remarkably, the concentrations showing activity were not toxic for primary human hepatocytes. Moreover, the platform developed provides a novel integrated high-throughput sustainable extraction method and readout to elucidate the mechanism of action of unknown compounds with respect to previous studies. • Plant compounds hold potential but applications are limited by low abundance and diversity. • Standardized growth and extraction are needed to unlock phytochemical potential. • Liver cirrhosis, a fatal condition, lacks a cure; global cases rise by 2039. • Hairy root cultures from plants used in high-throughput live-cell assays to study fibrosis. • Identified antifibrotic bioactivities of sustainably sourced plant extracts.