## 2 Results and Discussion
### Effect of Cannabis sativa L. Extracts on Viability of Hepatoblastoma HepG2 Cells  
The main anticancer effects of cannabinoids are attributed to the induction of the endocannabinoid receptors CB1 and CB2, which activate different signaling mechanisms leading to cell death by endoplasmic reticular stress and autophagy, apoptosis, as well as inhibition of cell proliferation [26]. CBD is the most promising cannabinoid for the cancer treatment, as it lacks the psychotomimetic properties of THC. The anti-proliferative and proapoptotic effects of CBD were shown on a variety of cancer types both in vitro and in mouse tumor models, where it was suggested to modulate the tumor microenvironment [16]. HepG2 cell line, a good in vitro model to toxicity studies also for cannabis and derivatives, was employed [27; 28]. Mary Moonlight-Legal Weed (Sample 1 in Table 3), Purple Rock-Legal Weed (Sample 5 in Table 3), and Alexis Haze-Legal Weed (Sample 20 in Table 3) extracts, with THC:CBD ratios of 1:9, were chosen as representative samples of cannabis light extracts to evaluate the effect on viability of hepatoblastoma cells HepG2 after 24, 48, and 72 h treatment. The three samples reported THC:CBD ratios of 1:9, optimal for inflammation and pain treatment. According to [PERSON] et al. [5], which suggested that cannabis extracts might show more potent anticancer activity than the pure substances due to the presence of other compounds, including flavonoids, terpenoids, sugars, and amino acids, we found that the three extracts had a comparable if not a more cytotoxic effect than CBD already after 24 h of treatment. The IC\({}_{50}\) were 14.35 \(\pm\) 3.68, 15.25 \(\pm\) 1.98 and 9.86 \(\pm\) 1.87 uM for Mary Moonlight-Legal Weed, Purple Rock-Legal Weed, and Alexis Haze-Legal Weed, respectively, and 16.82 \(\pm\) 2.54 uM for CBD (Figures 3 and 4 and Tables 4 and 5). The comparable activity could be related to their content of CBD equal to 3.53 (\(\pm\)0.09)% \(w\)/\(w\) and 3.7 (\(\pm\)0.2)% \(w\)/\(w\) for Mary Moonlight-Legal Weed and Alexis Haze-Legal Weed, respectively. Purple Rock-Legal Weed extracts containing 1.23 (\(\pm\)0.05)% \(w\)/\(w\) of CBD showed more cytotoxic effect. Up to 48 h, THC treatment did not show any toxic effect; at 72 h, it showed a greater effect (IC\({}_{50}\) 47.94 \(\pm\) 2.30 uM) than CBD (IC\({}_{50}\) 66.93 \(\pm\) 6.00 uM), and when used in combination, a synergic effect was observed (IC\({}_{50}\) 36.16 \(\pm\) 3.59 uM). The cell growth inhibitory activity of the three extracts did not change up to 72 h and remained higher than the pure substances. Figure 3: Dose–response curves of Mary Moonlight-Legal Weed, Alexis Haze-Legal Weed, and Purple Rock-Legal Weed extracts. HepG2 cells were grown for 24 h (**a**), 48 h (**b**), 72 h, and (**c**) in the presence of increasing concentrations of the three extracts. The viability of cells was evaluated using the MTT assay and the concentration required to reduce the cell number by 50% (IC\({}_{50}\)) for each condition was calculated using GraphPad Prism. Each data point represents the mean of at least three separate experiments and the vertical bars represent the S.D. \begin{table}
\begin{tabular}{c c c c} \hline
**Treatment** & **24 h** & **48 h** & **72 h** \\ \hline Mary Moonlight & 14.35 \(\pm\) 3.68 & 12.56 \(\pm\) 2.37 & 11.66 \(\pm\) 2.84 \\ [PERSON] & 15.25 \(\pm\) 1.98 & 12.99 \(\pm\) 2.84 & 11.48 \(\pm\) 2.39 \\ Purple Rock & 9.86 \(\pm\) 1.87 & 14.90 \(\pm\) 3.97 & 11.13 \(\pm\) 1.70 \\ \hline \end{tabular} Data are median inhibitory concentrations (IC\({}_{50}\)) values in uM \(\pm\) standard deviation value.  
\end{table} 
Table 4: IC\({}_{50}\) of [PERSON], Alexis Haze, and Purple Rock against HepG2 cells as determined by the MTT assay. Figure 4: Dose–response curves of THC, CBD, and MIX THC CBD. HepG2 cells were grown for 24 h (a), 48 h (b), 72 h, and (c) in the presence of increasing concentrations of the two cannabinoids, THC and CBD, either as single agents or in dual combinations prepared at a 1:9 ratio.