## 3 Results
### Cytostatic Effect of the Substances Present in the Most Active Sub-Fraction  
There are several components of M8R sample that can be responsible for the observed cytostatic effect. Stachydrine, the compound present in a higher concentration, had alone a weak inhibitory effect (Table 3) on the growth of HeLa cells (IC\({}_{50}\) = 70 \(\upmu\)M). Benzalkonium  
\begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline \multirow{2}{*}{**L b**} & \multicolumn{3}{c}{**Stachydrine**} & \multicolumn{3}{c}{**n-Alkane**} & \multicolumn{3}{c}{**Rutin**} & \multicolumn{3}{c}{**Rutin**} \\ \cline{2-10}  & \multicolumn{1}{c}{**13C**} & \multicolumn{3}{c}{**1H**} & \multicolumn{1}{c}{**13C**} & \multicolumn{1}{c}{**1H**} & \multicolumn{1}{c}{**13C**} & \multicolumn{1}{c}{**1H**} & \multicolumn{1}{c}{**13C**} & \multicolumn{1}{c}{**1H**} \\  & \multicolumn{1}{c}{**NMR**} & \multicolumn{1}{c}{**NMR**} & \multicolumn{1}{c}{**NMR**} & \multicolumn{1}{c}{**NMR**} & \multicolumn{1}{c}{**NMR**} & \multicolumn{1}{c}{**NMR**} & \multicolumn{1}{c}{**NMR**} & \multicolumn{1}{c}{**NMR**} & \multicolumn{1}{c}{**NMR**} \\ \hline
1 & & & & 13.1 & 0.90 & & & 1 & 101.0 & 5.10 \\
2 & 76.2 & & 4.01 & & 21.7 & 1.30 & 157.9 & nt & 2\({}^{\prime\prime}\) & 74.3 & 3.46 \\
3 & 25.2 & 2.30 & & 2.49 & 30.9 & 1.26 & 134.1 & nt & 3\({}^{\prime\prime}\) & 76.7 & 3.40 \\
4 & 18.4 & & 2.12 & & 28.4 & 1.25–12.8 & 178.0 & nt & 4\({}^{\prime\prime}\) & 70.0 & 3.25 \\
5 & 66.6 & 3.68 & & 3.49 & 28.8 & 1.25–12.9 & 157.0 & nt & 5\({}^{\prime\prime}\) & 75.8 & 3.31 \\
6 & 43.7 & & 3.13 & & 28.8 & 1.25–13.30 & 93.4 & 6.40 & 6\({}^{\prime\prime}\) & 67.1 & 3.37 \\
7 & 51.3 & & 3.30 & & 28.8 & 1.25–13.31 & 164.6 & nt & 1\({}^{\prime\prime}\) & 103.3 & 4.51 \\
8 & 169.4 & & - & & & 98.5 & 6.20 & 2\({}^{\prime\prime\prime}\) & 70.7 & 3.62 \\
9 & & & & & & 161.5 & nt & 3\({}^{\prime\prime\prime}\) & 70.8 & 3.52 \\
10 & & & & & & 104.2 & nt & 4\({}^{\prime\prime\prime}\) & 72.5 & 3.26 \\
1\({}^{\prime}\) & & & & & & 121.7 & nt & 5\({}^{\prime\prime\prime}\) & 68.3 & 3.43 \\
2\({}^{\prime}\) & & & & & & 116.2 & 7.66 & 6\({}^{\prime\prime\prime}\) & 16.5 & 1.10 \\
3\({}^{\prime}\) & & & & & & 144.4 & nt & & & \\
4\({}^{\prime}\) & & & & & & 148.3 & nt & & & \\
5\({}^{\prime}\) & & & & & & 114.6 & 6.87 & & & \\
6\({}^{\prime}\) & & & & & & 122.1 & 7.16 & & & \\ \hline \hline \end{tabular}  
* Chemical shift in \(\delta\) scale (ppm). b Location (structure in Figure 3). NMR spectra of M8R sub-fraction were obtained at 600 MHz in deuterated methanol (Met-44).  
\end{table}
Table 2: NMR spectroscopic data a for the compounds present in M8R sub-fraction isolated from _S. spinosum_.  
Figure 3: The chemical structure of the three major compounds presents in the M8R sub-fraction.  
chloride showed a strong growth suppressive effect against HeLa cells (IC\({}_{50}\) = 0.8 \(\upmu\)M) and MCF-7 cells (IC\({}_{50}\) = 5 \(\upmu\)M), but unfortunately also against normal cells (same intensity). The growth suppression of HeLa and MCF-7 cells by benzalkonium chloride at a concentration of 10 \(\upmu\)M was determined to be 88%, and 65%, respectively. The mixture of stachydrine with benzalkonium chloride at the same concentration of both compounds (5 \(\upmu\)M) completely suppressed the growth of all tested tumor cells (Jurkat, HeLa, MCF-7, and MDA cell lines). The same effect was observed for the mixture of 1 nM stachydrine and 5 \(\upmu\)M benzalkonium chloride (1:1) (Table 3).  
The IC\({}_{50}\) values of Paclitaxel as positive control on HeLa, MCF-7, and MDA cells were as followed: 4.2 \(\upmu\)M, 3.8 \(\upmu\)M, and 8.6 \(\upmu\)M, respectively.  
Rutin showed no inhibitory effect on cell growth of MCF-7 and HeLa at the concentrations of 10\({}^{-4}\) to 10\({}^{-9}\) M. Nevertheless, it showed a weak inhibitory effect against the growth of leukemic and mammary gland tumor cells.