Overexpression of ROSMARINIC ACID SYNTHASE enhances the hepatoprotective potential of Dracocephalum heterophyllum hairy root cultures

Dracocephalum heterophyllum Benth., a widely used Tibetan medicinal herb in the Lamiaceae family, accumulates bioactive secondary metabolites such as rosmarinic acid (RosA) and exhibits strong hepatoprotective, antioxidant, and anti-inflammatory activities. The sustainable production of this herb is limited by the extreme conditions required for its growth. To address this limitation, we developed an efficient axillary bud propagation system for D. heterophyllum , achieving a multiplication coefficient of 4.8. We then generated hairy roots from propagated shoot explants using Agrobacterium rhizogenes –mediated transformation. Among the tested A. rhizogenes strains, LBA9402 showed the highest induction efficiency on stem explants, reaching 23.3% ± 1.0. Heterologous expression of the C. blumei gene encoding rosmarinic acid synthase (RAS) in hairy roots resulted in a 1.2- to 2.1-fold increase in RosA levels compared with those in empty vector control roots. Consistent with the elevated RosA levels, the RAS- overexpressing hairy roots exhibited markedly enhanced lipid-lowering and hepatoprotective effects in oleic acid–induced HepG2 cells. These results establish bioengineered D. heterophyllum hairy root cultures as a scalable and sustainable source of bioactive compounds and as an alternative to wild-harvested plant material.