Bulgarian bilberry juice shows antioxidant and yeast-cell activity in laboratory study

Bilberry fruit collected in Bulgaria’s Rila Mountains contained 338.54 milligrams of anthocyanins per 100 grams of fresh fruit and displayed antioxidant activity in laboratory assays, according to a study that combined chemical analysis with tests in yeast cells. Malvidins and delphinidins were the dominant anthocyanin groups in the 2024 Belmeken sample, while the pressed juice showed strong radical-scavenging and reducing activity under the assay conditions. When added to yeast exposed to methyl methanesulfonate, a mutagenic chemical used to induce cellular injury, bilberry juice increased cell survival and reduced superoxide-anion levels.

These findings characterize the composition and experimental activity of one regional fruit sample; they do not establish clinical benefits in people. The investigation focused on European bilberry, Vaccinium myrtillus L., a small shrub that produces dark blue, juicy berries and grows in habitats including stony meadows, sparse coniferous forests and mountainous areas. It is widespread across Europe, Asia Minor, the Caucasus and Siberia, and its fruit has been used as food and in traditional medicine in Bulgaria and elsewhere.

The researchers collected fresh fruit in 2024 from the Belmeken region of the Rila Mountain. For measurements of total polyphenols, total flavonoids and antioxidant potential, the fruit was squeezed, and the resulting juice was filtered and frozen pending analysis. Detailed anthocyanin analysis used a separate set of five fruit samples.

Five grams of fruit were homogenized with 10 milliliters of 80% methanol, sonicated for 20 minutes and centrifuged at 10,000 times gravitational acceleration for five minutes. The extraction was repeated, the combined supernatants were evaporated, and the residue was redissolved in 5% methanol containing 1% formic acid. After solid-phase extraction, the investigators used ultra-performance liquid chromatography coupled to tandem mass spectrometry, or UPLC-MS/MS, to separate and identify anthocyanin forms.

Positive-ion electrospray ionization and multiple reaction monitoring were used to detect compounds, with high-energy collision dissociation at 45 volts providing the MS/MS transitions. Malvidins were the largest measured anthocyanin group, accounting for 38.08% of the total and measuring 128.9 ± 6.4 milligrams per 100 grams of fruit. Delphinidins followed at 33.15%, corresponding to 112.2 ± 5.6 milligrams per 100 grams.

Petunidins were present at 63.0 ± 3.1 milligrams per 100 grams and represented 18.6%, while cyanidins measured 34.4 ± 1.7 milligrams per 100 grams and represented 10.1%. The sum of the quantified groups gave the reported total anthocyanin content of 338.54 milligrams per 100 grams of fresh fruit. The juice was also assessed with broader colorimetric tests.

Total polyphenols measured 8.40 ± 0.29 milligrams of gallic acid equivalents per 100 milliliters, with the Folin-Ciocalteu reagent method used for this determination. Total flavonoids were 7.26 ± 0.11 milligrams of quercetin equivalents per 100 milliliters, based on an aluminum chloride colorimetric assay. Each measurement was performed at least three times, and the reported values are means with standard deviations.

These assays quantify broad classes of compounds expressed in reference-equivalent units rather than directly identifying every constituent present in the juice. Three complementary tests indicated substantial antioxidant potential in the tested sample. In the DPPH assay, it achieved 89.0 ± 2.1% scavenging.

The ferric reducing antioxidant power assay yielded 2.4 ± 0.09 millimoles of Fe(II) per liter. These results were obtained under defined laboratory conditions, where each method measured a particular form of radical scavenging or chemical reducing capacity. They therefore demonstrate antioxidant activity of the juice in those systems, not an improvement in health outcomes or oxidative-stress biomarkers in people.

The researchers next examined the juice in Saccharomyces cerevisiae. They treated exponential-phase cell suspensions for 30 minutes with bilberry juice at either 1% or 10%, using methyl methanesulfonate, abbreviated MMS, as a positive control for cellular injury. Juice alone did not reduce cell survival relative to untreated cells, indicating that the tested concentrations were not detectably cytotoxic within this experimental design.

When juice was combined with MMS, both concentrations produced significant cytoprotection, described as a 1.5-fold increase in cell survival with P < 0.0001. Survival in the combined-treatment groups was similar to control levels, and no statistically significant difference was detected between the 1% and 10% treatments. The cell-based analysis also measured physiologically active superoxide anions through an XTT-based procedure in which the compound enters living cells and is reduced by superoxide anions to colored formazans.

Bilberry juice alone did not induce reactive oxygen species relative to untreated cells, and the measured levels were comparable with control values. In the presence of MMS, however, both juice concentrations reduced the induced superoxide-anion signal by approximately fivefold. Levels in the combined treatments were described as indistinguishable from those of untreated cells, with no detectable advantage observed when the juice concentration increased from 1% to 10%.

Under this specific yeast model, the results therefore showed two linked observations: protection of cell survival and suppression of MMS-associated superoxide generation. The anthocyanin concentration measured in the Belmeken fruit exceeded values previously reported for two other Bulgarian locations. Earlier work cited by the investigators reported 217 milligrams per 100 grams for fruit from Velingrad and 162.4 milligrams per 100 grams for fruit from Troyan, compared with 338.54 milligrams per 100 grams in the present sample.

The Belmeken result was also higher than the cited values of 143.9 milligrams per 100 grams for a commercially available bilberry juice and 297.4 milligrams per 100 grams in another previous measurement. The authors identify regional, climatic and year-to-year factors as possible contributors to such variation, but the present comparisons were not controlled experiments designed to isolate those influences. The data show a higher concentration in the analyzed sample than in the cited reports, not a general or time-based ranking among Bulgarian sites.

Earlier studies reviewed in the paper provide experimental context while remaining distinct from the current findings. Bilberry extracts have reduced markers of oxidative damage in cultured skin and neuronal cells, and anthocyanin-rich extracts have protected mice from stress-induced liver injury in animal experiments. The paper also notes that human ingestion studies have not consistently found significant changes in biomarkers of antioxidant status or oxidative stress.

It points to variations in extract standardization, anthocyanin concentration and redox conditions as possible considerations, while also noting that effects attributed to antioxidant phytochemicals may be indirect or independent of antioxidant activity. These distinctions matter because the present work used fruit-derived juice, a single collection location, chemical assays and a yeast-cell model rather than a standardized clinical trial in humans. Anthocyanins are also vulnerable to degradation during industrial processing, which can affect the color and properties of bilberry-derived products.

The study’s combined measurements show that the Belmeken fruit contained a substantial and chemically differentiated anthocyanin reservoir, and the yeast experiments found biological activity under controlled conditions. The research did not test how processing, storage or passage through the digestive system changes the composition or effects of the juice, nor did it compare the clinical relevance of the two tested concentrations. Its support for Bulgarian bilberry as a source of anthocyanin-rich material therefore rests on the documented chemical profile, the laboratory antioxidant assays and the cytoprotective results in yeast.

More standardized and appropriately designed human studies would be required before any conclusion about therapeutic effects or disease prevention could be drawn.

Subject of Research: Chemical composition, antioxidant assays, and yeast-cell effects of Bulgarian bilberry juice

Article Title: Phytochemical composition and biological activity of bilberry fruit juice ( Vaccinium myrtillus L.) from Bulgaria

Article References: Kiselova-Kaneva, Y., Dimitrov, Z., Ignatov, I., Popova, T., Gotova, I., Todorova, T., Boyadzhiev, K., Angushev, I., & Ivanova, D. (2026). Phytochemical composition and biological activity of bilberry fruit juice (Vaccinium myrtillus L.) from Bulgaria. Pharmacia, 73, Article e188976. https://doi.org/10.3897/pharmacia.73.e188976

Image Credits: AI Generated

DOI: 10.3897/pharmacia.73.e188976

Keywords: bilberry, Vaccinium myrtillus, anthocyanins, polyphenols, antioxidant assays, Bulgaria, Rila Mountains, yeast cells, oxidative stress, UPLC-MS/MS, Phytochemical, composition