Table 2.

Colorectal cancer: Main results of in vitro studies.

Study	Berry	Cell Line	Endpoints	Methods	Exposure	Effect
Katsube et al., 2003 [41]	Bilberry, lingonberry	HCT116	1. Cell growth inhibition 2. Induction of apoptosis (for bilberry only)	1.Cell counting by trypan blue exclusion 2. DNA extraction and agarose gel electrophoresis/Analysis of nuclear morphology by 1 mM bisbenzimide staining	1. 2–4 mg dry weight/mL of bilberry/lingonberry ethanol extract for 48 h, in addition bilberry ethanol extract for 24 and 48 h with 0.5–4 mg dry weight/mL 2. 4 mg/mL bilberry extract for 24 h	1. Bilberry and lingonberry ethanol extracts strongly inhibited the growth of HCT116. Bilberry inhibited cell growth >50% with 2–4 mg/mL. Over 50% inhibition was achieved also with 4 mg/mL lingonberry extract 2. Bilberry extract did not induce apoptosis
Olsson et al., 2004 [42]	Lingonberry	HT-29	Cell growth inhibition	WST-1 assay	Lingonberry ethanol extract/anthocyanin fraction 0.025–0.5% of plant dry matter of total weight in the wells for 24 h	Decreased proliferation significantly and dose-dependently. GI50 between 0.25% and 0.5% of plant dry matter in the wells Anthocyanin fraction was less effective
Zhao et al., 2004 [43]	Bilberry	HT-29 NCNM460 (Immorta-lized colon cell line)	Cell growth inhibition	Sulphorhodamine B assay	Bilberry anthocyanin-rich extract (Artemis International Inc.) 25–75 µg of monomeric anthocyanin/mL for 24/48/72 h	Significantly and time-dependently inhibited growth of HT-29 with 50–75 µg/mL of monomeric anthocyanin from 24 h on, with 25 µg/mL from 48 h on. Over 50% inhibition for all concentrations after 72 h. NCNM460 was only inhibited after 72 h
Wu et al., 2007 [44]	Bilberry, lingonberry	HT-29	1. Cell growth inhibition 2.Induction of apoptosis	1.Total cell count determined using SYTOX-green 2.DNA fragmentation by agarose gel electrophoresis	1.5–60 mg/mL bilberry/lingonberry methanol extract for 24 h 2. 5–60 mg/mL bilberry extract for 48 h	1. Both inhibited cell growth significantly. Bilberry GI50 = approx. 15 mg/mL Lingonberry GI50 = 60 mg/mL 2. Bilberry induced apoptosis with 20–60 mg/mL
Jing et al., 2008 [45]	Bilberry	HT-29	Cell growth inhibition	Sulphorhodamine B assay	Bilberry anthocyanin-rich extract (Artemis International, Inc.) 0–200 µg/mL cyanidin-3-glucoside equivalents for 48 h	Dose-dependent inhibitory effect on the growth of HT29. GI50 = 32.2 µg/mL
McDougall et al., 2008 [46]	Lingonberry	Caco-2	Cell growth inhibition	Dojindo CCK-8 kit	Lingonberry acetonitrile extract (bound fraction from solid phase extraction only) 25–75 µg of gallic acid equivalents/mL for 72 h	Inhibited cell growth in a dose-dependent manner, GI50 38.3 µg GAE/mL
Schantz et al., 2010 [52]	Bilberry	HT-29 Caco-2	1.Cytotoxicity 2. Induced DNA damage	1. Alamar blue 2. Comet Assay	Bilberry extract from European bilberry pomace (Kaden Biochemicals, Hamburg, Germany) 1. 5–500 µg/mL for 1 and 24 h 2. 0.01–500 µg/mL for 1 and 24 h	1.Significant effect only on HT-29, with 500 µg/mL only. EC50 was not reached 2. Decreased induced DNA damage only in Caco-2 cells, with 5–100 µg/ml
Esselen et al., 2011 [47]	Bilberry	HT-29	1. Cell growth inhibition 2. DNA integrity 3. Drug interaction with topoisomerase poisons	1. Sulphorhodamine B assay 2. Comet assay 3. ICE assay with topoisomerase poisons Camptotecin and Doxorubicin	Bilberry industrial ARE (Indena, Milan, Italy) 1. 50–500 µg/mL for 72 h 2. 1–0 µg/mL for 1 h 3. 0.01–50 µg/mL, 30 min pre + 1 h coincubation	1. Bilberry ARE inhibited HT29 growth dose-dependently. GI50 was not reached 2. Bilberry ARE increased the rate of DNA strand brakes, but no additional oxidative damage observed 3.DNA-damaging effects and cytotoxicity of both drugs were inhibited by bilberry ARE
Fan et al., 2011 [48]	Lingonberry	HT-29	Cell growth inhibition	MTS assay	Lingonberry acetone extract 20–80 mg/mL for 48 h	Proliferation significantly inhibited in a dose-dependent manner, GI50 approx. 35 mg/mL
Kropat et al., 2013 [50]	Bilberry	HT-29	Cell growth inhibition	Living cells counted after staining with trypan blue	Pomace methanol extract from European bilberry 10–400 µg/mL for 72 h	Inhibited cell growth significantly, with concentrations above 100 µg/mL. GI50 between 200 and 400 µg/mL
Aaby et al., 2013 [49]	Bilberry	Caco-2 HT-29 HCT116	1. Cell growth inhibition/viability 2. Apoptosis (HT-29 only)	1. MTT Assay 2. Cell Death Detection ELISAPLUS Assay with BCA protein assay	Bilberry extract, raw juice and press residue extracts obtained from extraction in different temperatures (40/60/80/100 C) 1. 30 min exposure to 75–250 mg GAE/l. Measurement after 24 h 2. 24-h exposure with 75–250 mg GAE/l of press residue extracts	1. All extracts inhibited proliferation of all cell lines. Dose-response inhibition of all extracts on all cell lines, except for raw juice on HT-29 and HCT 116. GI50 of Caco-2 and HCT 116 was reached with all extracts, of HT-29 only press residue extracts from 80–100 °C temperatures 2. Dose response for both tested press residue extracts (extracted in 40/100 ºC temperature)
Brown et al., 2014 [53]	Lingonberry	HT-29 HT115	1. Cytotoxic activity 2. Inhibition of invasion and migration	1. MTT assay 2. Matrigel invasion and migration assays	Lingonberry methanol extract prepared to: IVDL: In vitro digested extract and IVFL: In vitro fermented extract both 3–50 µg/mL GAE for 24 h Ileal fluid after lingonberry ingestion, 3–200 µg/mL for 24 h	1. No cytotoxic activity for any exposure 2. IVDL and IVFL had significant anti-invasive effects, migration not affected
Šavikin et al., 2014 [54]	Bilberry	LS147	Cell viability	MTT assay	Bilberry decoction tea, bilberry infusion tea 12.5–200 µg/mL for 72 h	Both teas decreased viability Decoction tea EC50: 176.32 µg/mL Infusion tea EC50: 178.52 µg/mL
Tumbas Šaponjac et al., 2014 [51]	Bilberry	HT-29	Cell growth inhibition	Sulphorhodamine B assay	Bilberry extract fractions Fraction 1: Polar substances Fraction 2: 6 flavonols detected Fraction3: 8 phenolic acids identified 62.5–1000 µg of dried fraction/mL for 48 h	Fractions 2 and 3 suppressed cell growth significantly and dose-dependently. Over 50% growth inhibition achieved with ≥125 µg/mL of fraction 2, and with ≥500 µg/mL of fraction 3. Fraction 1 resulted in less than 10% inhibition with 250–500 µg/mL.
Minker et al., 2015 [55]	Bilberry, lingonberry	SW840 (primary) SW620 (metastasis)	Induction of apoptosis	Flow cytometry, cell surface phosphatidylserine detection, caspase 8 and caspase 9 activation	Bilberry/lingonberry proanthocyanidins extracted in acetone/methanol 5–75 µg/mL of each fraction during seeding and for 24 h for SW620 and 48 h for SW480	Bilberry induced apoptosis via extrinsic pathway EC50 for bilberry treated SW620: 24.7 µg/mL and SW480: 25.2 µg/mL EC50 for lingonberry treated SW620: 24.3 µg/mL SW480: 24.7 µg/mL
Borowiec et al., 2016 [56]	Bilberry	Caco-2	1. Cell viability 2. Genotoxicity	1. MTT assay 2. Single cell electrophoresis, also under oxidative stress (H2O2)	Bilberry juice extract (no solvent used) 1. 12.5–400 µg dry mass/mL for 48 h 2. 100 µg dry mass/mL 48 h	1. Viability of Caco-2 was significantly but modestly inhibited only with 400 µg/mL (approx. 20% inhibition) 2. Not genotoxic
Mudd et al., 2020 [39]	Bilberry	HCT 116 HT-29 CCD-18Co (Human colon)	Inhibition of proliferation/Cell viability	MTT assay	Bilberry anthocyanin extract concentrations up to 200 µmol/L	Bilberry anthocyanin extract inhibited proliferation of tumor cells more than colon cells IC50 values for HT-29: 124 µmol/L HCT-116: 75 µmol/L CCD-18Co: 1050 µmol/L
Vilkickyte et al., 2020 [40]	Lingonberry	HT-29	Cell viability	MTT assay	Lingonberry extracted in acetone and phenolic fractions subsequently isolated with column chromatography	Lingonberry extract fractions reduced viability with EC50 values approx. 0.05–1.1 mg/mL, Fraction 4 rich in proanthocyanidins being the most effective