# Comparative Study of _Plantago media_ Extracts in the Treatment of _Acanthamoeba_ sp. Trophozoites
## 3 Results  
The protocol of efficient micropropagation of _Plantago media_ L. was developed previously [19]. The shoots and roots of in vitro micropropagated plantlets were chosen for the experiments to compare whether raw materials produced by biotechnological methods are richer in selected secondary metabolites and have higher biological activity, in this case, acanthamoeba. Moreover, to compare the activity of extracts resulting from the presence and content of bioactive compounds, _Plantago media_ was compared to the well-known and valued raw material of _Plantago lanceolatae folium_ (Figure 1). Figure 1: Plant materials used for the investigations: _Plantago media_ in soil cultivation (**a**) with zoomed infloresances (**d**) and leaves (**e**); and _P. media_ in-vitro-derived leaves (**b**) and roots (**c**). Chromatographic examination of extracts by 2D-TLC fingerprinting on cellulose plates (Table 1) exhibited a similar pattern of phenolic compounds in shoots and roots of the micropropagated plantlets. Phenylethanol glycosides, such as acteoside and plantamajoside, were present, and no flavonoids could be detected. The phenylethanoloids appeared under UV light as blue bands exhibiting strong blue fluorescence after spraying with NA. In turn, no yellow or brown bands changing color to brilliant yellow fluorescence under UV upon derivatization with aluminum chloride, characteristic for flavonoids, were observed. The flavonoids were apparent in the extracts of intact plants--_P. media_ inflorescences and leaves and _P. lanccolata_ leaves (Table 1). \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Material (Water Fraction of an Extract)**} & \multicolumn{2}{c}{**2D-TLC Cellulose**} & \multirow{2}{*}{**Flavonoids**} & \multirow{2}{*}{**Phenylethanoloid Glycoses**} \\  & **(Adsoorbent: Cellulose; Mobile Phases:** & & \\  & **I: BAW 4:15, II: 15\%AcOH;** & & \\  & **Detection—UV 365 nm)** & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: TLC-fingerprinting analysis of phenolic compounds in _Plantago media_. The results of quantitative analyses performed using the HPLC-DAD method indicate differences in the content of individual compounds--phenylethanolanoids, flavonoids and phenolic acids--depending on the type of _Plantago media_ L. raw material (Table 2). The leaves of plants from the ground and from in vitro cultures possess the highest content of the sum of phenylethanolanoids as well as individual compounds--atecoside and isoaceoside. Moreover, the content of acetoside is more than two-fold higher in the leaves of the plant from the ground (6641.70 \(\pm\) 12.06 mg/100 g DW) than from the shoot culture (3034.11 \(\pm\) 15.54 mg/100 g DW). Roots from in vitro cultures contain lower contents of acetoside (1039.31 \(\pm\) 9.18 mg/100 g) and isoaceoside (161.93 \(\pm\) 6.42 mg/100 g), although more than infloreserences (655.13 \(\pm\) 5.16 mg/100 g DW and 67.68 \(\pm\) 2.50 mg/100 g DW, respectively), which are the least rich in compounds, both phenylethanolanoids and phenolic acids. In general, it can be observed that acetoside is the dominant compound in each of the raw materials of this species, regardless of the type of organ (leaf, root, or inflorescence) or origin (organs of natural plants or biomass from biotechnological production). Acteoside and isoaceoside (phenylethanolanoids) are present in raw materials in a much higher concentration than the sum of phenolic acids or the sum of flavonoids. The content of acetoside constitutes 87.76% and 84.15% (in vitro shoots and roots), 84.96% and 93.83% (in _P. media_ infloresences and leaves), and 93.60% (in _P. lanccolata_ leaves) of the phenolics sum content. For comparison, the content of these compounds in _Plantaginis lanccolatae__folium_, a well-known pharmacopoeial raw material, was also examined and it turned out that the leaves of _P. media_ are richer in acetoside and isoaceoside than the leaves of _P. lanccolata_ (Table 2).