Different concentrations of TiO₂ NPs (0, 10, 20, 30, and 50) were prepared for hairy root treatments. 0.5 g of .S. officinalis hairy roots were transferred to 250 mL Erlenmeyer flasks containing 15 mL of liquid MS culture medium with three replicates. Then, they were placed in an incubator shaker at 110 rpm and 25 &#8451 in dark conditions. On the 22nd day, the liquid MS culture media containing different concentrations of nano titanium dioxide was added to Erlenmeyer flasks. 24 and 48 h after treatment, the hairy roots were taken out and transferred to the MS culture medium lacking elicitor.

The highest rate of total phenol (9.79 mg GLA/g FW) and total flavonoid contents (1.06 mg QE/g FW) were obtained in the treated hairy roots with 50 and 30 mg/L of nano elicitor in 24 and 48 h of treatments, respectively. The maximum level of most polyphenols, such as rosmarinic acid, cinnamic acid, and rutin, was produced in 24 h of treatment. The use of TiO₂ NP for 48 h with 50 mg/L concentration showed the highest production level of SO6 protein.