# Biocompounds and Bioactivities of Selected Greek Boraginaceae Seeds
## 4 Discussion
### Antiproliferative Activity of the Water/Methanol Seed Extracts on HT-29 Cancer Cells  
A colon cancer human cell line was selected for evaluating the antiproliferative activity of the phenolic-containing extracts because colorectal cancer is one of the most common cancers worldwide and the fourth leading cause of death, with mortality close to 50% in Western countries. Also, several studies suggested that it is a disease strongly influenced by diet, which can modulate inflammatory processes and affect gene expression in the body [7].  
The effects of the seed extracts of selected taxa on HT-29 cell viability are shown in Figure 2. After 48 and 72 h of treatment, the MTT assay revealed concentration- and time-dependent inhibitory effects on HT-29 cells mediated by most seed extracts. The extracts of _C. major_ and _L. zalnui_ induced the greatest decrease in cell viability at 72 h at the maximum concentration tested (1000 \(\upmu\)g/mL), which was \(\sim\)5-30% lower than that obtained at 48 h. The doses of extracts that inhibited cell growth by 50% (GI\({}_{50}\)), as well as those of some pure phenolic compounds occurring in the checked extracts, are depicted in Figure 3. GI\({}_{50}\) values after a 72-h treatment period for _L. zalnui_, _C. major_, _S. creticum_, _O. frutescens_, and _A. gracca_ were 375, 600, 880, 900, and 990 \(\upmu\)g/mL. These results are in good agreement with those obtained for the phenolic-containing seed extracts from several Borago_ species [23] and _Buglossoides_ taxa [14]. Finding inhibitory effects against HT-29 cells exercised by the phenolic extract of any Boraginaceae seed is noteworthy, given that such cells tend to be unresponsive to phenolics. For instance, the phenolics extracts from another GLA species, _Oenothera paradoxa_, induced apoptosis in Caco-2 cells, while HT-29 cells were slightly affected [52].  
The antiproliferative activity against HT-29 cells of pure phenols occurring in the studied extracts was also checked: rosmarinic acid, quercetin, cafeic acid, rutin, and chlorogenic acid. These showed GI\({}_{50}\) of 35, 76, 500, 680, and 680 \(\mu\)g/mL. Interestingly, the species that displayed higher antitumor activity, i.e., _C. major_, contained high amounts of rosmarinic acid, which is the more active phenolic against HT-29 cells; thus, this activity was probably influenced partially by this phenolic.  
Finally, the SI of HT-29 vs. normal cells (CCD-18) was calculated for the more active extract, i.e., _L. zahtii_ (see Materials and Methods-Section 2). Extracts having SI > 2 are classified as highly selective against cancer cells, while the ones with SI < 2 exhibited toxicity to normal cells [53]. For research on biological extracts and/or isolated compounds, this figure is critical for deciding further research on the subject [54]. Although data were favorable, this research should be continued with appropriate experiments before considering this extract as an anticancer drug candidate. This is mandatory, because although in vitro drug/extract dose-response experiments constitute the cornerstone of the preclinical evaluation of anticancer drugs, the transition from in vitro anticancer activity to clinical application needs careful analysis and validation of results.  
It is timely to indicate that in the case of trying to obtain oils exhibiting antitumor bioactivity from the analyzed Greek Boraginaceae taxa, it would be necessary to process the seeds by cold pressing. This is so because oils extracted through organic solvent use, e.g., _n_-hexane, lack phenolic compounds, given the hydrophilic nature of the latter.