## 3 Results and Discussion
### Antiproliferative Activity of the Water/Methanol Seed Extracts on HT-29 Cancer Cells
The Selectivity Index (SI) for 72 h exposed cells to seed extracts is shown in parentheses over the columns. After 48 and 72 h of treatment, the MTT test revealed time- and concentration-dependent inhibitory effects on HT-29 cells for all assayed extracts. Cell viability at 48 h at the maximum concentration tested (300 \(\upmu\)g/mL) and for the different species was \(\sim\)20% lower than that obtained at 72 h. After 72 h of culture, cell growth inhibition was exerted much better by the seed extracts of _R. nigrum_ 'Koksa' and _Ribes_ 'Erkeeni', which show a GI50 value of 37 and 42 \(\upmu\)g/mL. According to the threshold proposed by [PERSON] and [PERSON] [32], crude extracts showing a GI50 \(\leq\) 100 \(\upmu\)g/mL can be considered to be cytotoxic and selected for further studies, whereas the most promising ones are those with a GI50 lower than 30 \(\upmu\)g/mL. Thus, all seed extracts whose GI50 is detailed in Figure 3 merit further research for their fractionation until pure active compounds are isolated. After that, the mechanisms of action of such compounds against various cancer cell lines would be established according to proper experimentation. All assayed _Ribes_ extracts, except _R. nigrum_ 'Hara katarlik', showed activity higher than the _R. rubrum_ extract (GI50 at 72 h of cell exposure to the extract of 99 \(\upmu\)g/mL). The extracts of _R. nigrum_ 'Hara katarlik' showed an undetermined GI50 value higher than 300 \(\upmu\)g/mL. Interestingly, the highly bioactive extract from _Ribes_ 'Erkeeni' contains all detected phenolics, while _R. nigrum_ L. 'Koksa' lacks only populin. Therefore, the high bioactivity found for such extracts could be due to a synergy between all the compounds detected. However, it needs to be considered that the polysaccharide-rich fraction in _R. nigrum_ has been characterized as highly bioactive [33], and that the water/methanol extracts obtained from the seeds assayed in this work can include some amounts of polysaccharides; thus, a synergy between phenolic compounds and seed polysaccharides could be also responsible for the noted antitumor effects against HT-29 cells, especially considering that these cells are not too sensitive to phenolic compounds [11; 34]. Finally, the selectivity index (SI) of HT-29 versus normal cells CCD-18 was calculated (see Material and Methods, Section 2). An extract with an SI value greater than two is considered as high selectivity against cancer cells, whereas one with an SI value less than two demonstrates general toxicity to normal cells [14]. Such value for any research on herbal drugs and/or isolated compounds is critical for determining whether further research can be continued [35]. The SI at 72 h ranged from 17 (_Ribes_ 'Myuryuchenee') to 32 (_Ribes_ 'Erkeeni'), from which it can be concluded that the seed extracts of _Ribes_ have a high selectivity against HT-29 human colorectal cancer cells. However, the data exposed here should be complemented with further experiments before considering the extracts checked in this work as anticancer drug candidates. This is because although the measurement of drug/extract dose-response in cultured cells is the cornerstone of the preclinical assessment of anticancer drugs, the transition from in vitro antitumor activity to clinical relevance requires cautious interpretation and further validation. Figure 2: MTT assay. A: concentration–response plot for HT-29 cells after exposure to seed extracts for 48 (**A**) and 72 h (**B**). Data represent the mean of three complete independent experiments \(\pm\) SD (error bars) with statistical significance equal to \({}^{*}\)\(p\)\(<\) 0.05; \({}^{**}\)\(p\)\(<\) 0.01.