## 2 Results
### Effect of Carvacrol Treatment on Ginsenoside Production in Hairy Roots of P. quinquefolium  
In hairy root cultures of _P. quinquefolium_ cultivated in shaken flasks, the total content of ginsenosides and the levels of protopnaxadiol and protopanaxatriol derivatives were estimated after 24 h and 72 h of treatment with different carvacrol concentrations (5, 10, 25,  
Figure 3: Normalized expression of _FPS_, _SS_, and _DS_ genes in hairy root cultures of _P. quinquefolium_ elicited with carvacrol. 50, 100, 250, and 500 \(\upmu\)M). Moreover, the level of eight individual metabolites--Rb1, Rb2, Rb3, Rc, Rd, Rg1, Rg2, and Re--was examined. This study showed that carvacrol induced changes in the total content of saponins depending on the time of exposure and the concentration of carvacrol. A shorter elicitation (24 h) with carvacrol at 5-50 \(\upmu\)M resulted in a slight increase (by about 10%) in ginseng content compared to the control samples (Figure 4). In contrast, longer exposure with carvacrol at 5-50 \(\upmu\)M resulted in a slight decrease (also by about 10%) in saponin accumulation. The highest level of these metabolites was observed with 100 \(\upmu\)M of carvacrol for both times of treatment, and its values were 19.75 and 20.01 mg\(\cdot\)g\({}^{-1}\) d.w. (no statistically significant differences), respectively, after 24 and 72 h of elicitation. An amount of carvacrol higher than 100 \(\upmu\)M reduced the saponin level for both times of elicitor treatment. At a 250 \(\upmu\)M concentration of carvacrol, it was observed that the decrease was more intense after an extended time of exposure (72 h). The highest amount of carvacrol (500 \(\upmu\)M) dramatically resulted in the depletion of the total saponins, regardless of the elicitation time. Then, their level was significantly lower than that determined in untreated samples. The results of the experiments also showed that ginseng belonging to the Rb and Rg groups responded differently to carvacrol treatment. The accumulation of protopanaxadiol derivatives (expressed as the sum of Rb1, Rb2, Rb3, Rc, and Rd) was inhibited after carvacrol treatment, regardless of the concentration of the elicitor and the time exposure, except for 5 \(\upmu\)M of carvacrol after 24 h of treatment. In contrast, the level of protopanaxatriol derivatives gradually increased with an increasing carvacrol concentration, until the maximum level was reached at 100 \(\upmu\)M of carvacrol, and then the ginseng content gradually decreased to the level observed in the control. The highest content of protopanaxatriol (expressed as the sum of Re, Rg1, and Rg2) was achieved after 72 h of exposure (100 \(\upmu\)M of carvacrol). The level was six-fold higher than that in untreated samples. An estimation of the ratio of derivatives belonging to Rb-group ginsengides and Rg-group ginsengides (RbG/RgG) allows for the assessment of the quality of ginseng root. The obtained results (Figure 5) showed that this parameter was the highest (5.56) in the control samples. Its gradual reduction to 0.66 was found with an increasing carvacrol  
Figure 4: The content of ginsengides depending on carvacrol concentration in _P. quinquefolium_ hairy root cultures, “c” means control sample. concentration after a shorter time exposure. The lowest level of RbG/RgG (0.59) was noted after 72 h of treatment with 100 uM of carvacrol, and it was more than nine-fold lower than the level in untreated samples. The observed changes in RbG/RgG levels indicated that the protopanaxatriol derivative content significantly increased after carvacrol elicitation. This research also determined the content of individual saponins and the profile of their change depended on the condition of carvacrol elicitation (Figure 6). Saponins Rc and Rb2 increased their level, respectively, by about 20% and 46% after 24 h of exposure with 5 uM of carvacrol. A higher concentration of carvacrol and a longer time of elicitation reduced the Rc and Rb2 content. Other results were noted in relation to compounds Rb1 and Rd.