1. Bioactive potential of lectin-enriched protein fractions from Thymus vulgaris L. and Urtica dioica L. grass V. Bakšenskaitė 1, *, L. Bistrovaitė 2, G. Balčiūnaitė 3, V. Kavaliauskis 3, V. Bendokas 4, V. Stanys 4, D. Baniulis 4, D. Majienė 2, J. Liobikas 2, J. Bernatonienė 3, A. Savickas 3 1 Department of Biochemistry and Biotechnologies, Faculty of Natural Sciences, Vytautas Magnus University, Vileikos 8, Kaunas, Lithuania; 2 Institute of Neurosciences, Medical Academy, Lithuanian University of Health Sciences, Eiveniu 4, Kaunas, Lithuania; 3 Department of Drug Technology and Social Pharmacy, Faculty of Pharmacy, Medical Academy, Lithuanian University of Health Sciences, Mickeviciaus 9, Kaunas, Lithuania; 4 Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry, Babtai, Kaunas reg., Lithuania Abstract. Medical herbs and spice plants present a valuable source for manufacturing biopharmaceutical medicines, and substantial amounts of the herbs are consumed for food worldwide. Herb production is established as one of priority areas for non-traditional agriculture in Lithuania. Lamiaceae family of plants include a number of valuable medical and spice plants that are represented by wild species or being common feature of gardens and agricultural production in Lithuania. Common thyme (Thymus vulgaris L.) is valued for the rich content of etheric oils, vitamins and minerals, and it is popular in food industry as a spice. Stinging nettle (Urtica dioica L.), a member of Urticaceae family, is another plant common in Lithuania and has an established medical value. Although content of mineral and organic substances of the medical herbs is well characterized, presence of bioactive polypeptides is much less appreciated. Lectins (phytohemagglutinins) are glycoproteins common in plants that demonstrate a broad spectrum of bioactive effects. Recent investigations revealed a potential for use of lectins as anticancer agents. Meanwhile mainly leaves or stems are consumed in case of a number of popular medical and spice herbs, including the nettle and thyme, seeds and roots of plants have been established as a common source for isolation of lectins from plants, so far. Therefore data on the presence of lectins in the leaves or stems of the two herbs is ambiguous. In this study, we prepare lectin-enriched protein fractions from extracts of the two herbs and assess their blood cell agglutinating activity using tests employing rabbit erythrocytes and macrophage J774 cells. The study revealed that lectin-like glycoprotein-enriched fractions from Common thyme and Stinging nettle grass contained specific hemagglutination activity up to 1.3 and 3.0 mg protein/ml, respectively. Application of the fractions containing the hemagglutination activity induced aggregation of macrophage J774 cells, and a highest cell death induction was observed in case of extracts obtained from herb of Common thyme. The results suggest presence of substantial hemagglutination activity content in grass of the two common medical herbs and bioactive potential of the lectin proteins. References: Bradford, M. (1976) Anal. Biochem. 72: 248-254; Franco-Fraguas, L., et al. (2003) Braz. J. Med. Biol. Res. 36(4) 447-457. Acknowledgment: The work was supported by the Sciences and Research Council of Lithuania grant No. MIP-10180. Fig. 1. Viability of macrophage J774 cell culture after treatment with lectin from Solanum tuberosum, and protein fractions of Thymus vulgaris and Urtica dioica. Table 1. Characteristics of protein fractions from Urtica dioica herb and extract. Urtica dioica L. Table 2. Characteristics of protein fractions from Thymus vulgaris extract. Methods. 1 Specific hemagglutinating activity was defined as the ratio of the titer/ml and protein concentration (mg/ml); Titer per ml was defined as the reciprocal of the highest dilution giving visible agglutination of the rabbit erythrocytes. 2 Lectin content is based on cell agglutinating activity as compared to specific hemagglutinating activity of lectin from Solanum tuberosum. Tables includerepresentative results from at least two separate experiments. Abbreviations: AS, ammonium sulphate. Powder of pulverized herb or dried extract was extracted for two hours with 5 volumes of 10 mM phosphate-buffered saline (PBS: 7.0 mM Na 2 HPO 4, 2.7 mM NaH 2 PO 4, 154.0 mM NaCl, pH 7.2) containing protease inhibitors. Cleared extract was subjected to 40% saturated ammonium sulphate precipitation for 2 h. The supernatant obtained after centrifugation at 15,000 ×g for 15 min was again saturated to 80% with ammonium sulphate for 24 h. Ammonium sulphate-precipitate was collected by centrifugation. The ammonium sulphate pellets were dissolved in PBS buffer and dialysed extensively against the same buffer. Total protein concentration was determined using method described by Bradford [1976]. Hemagglutination assay was carried out using trypsin treated rabbit erythrocytes as described by [Franco-Fraguas et al., 2003]. Macrophage J774 cell culture was maintained in Dulbecco’s modified Eagle’s medium with supplements at 37 o C and 5% CO 2. For bioactivity tests, cell culture medium was supplemented with the protein fractions of herb extracts in PBS containing 0,84-0,86 mg/ml specific hemagglutination activity and incubated for 24 h. Conclusions:  Presence of hemagglutination activity was observed for all of the protein fractions isolated by salting out with ammonium sulphate from fresh and dried grass of U. dioica and dried extracts of the herb. The highest yield of protein and specific hemagglutination activity were observed for protein fractions isolated from fresh grass. Lectin content estimated based on functional activity using lectin from Solanum tuberosum as a standard varied from 0.01 to 0.33 %. The highest lectin content was present in protein fractions isolated from the dried extracts.  For protein fractions isolated from extracts of Th. vulgaris, protein yields and specific hemagglutination activity was comparable to similar protein preparations from U. dioica. However, hemagglutination activity was not detected in 80 % saturated ammonium sulphate fraction. Lectin content was estimated at 0.02-0.03 %.  Application of the fractions containing the hemagglutination activity induced aggregation of macrophage J774 cells, and a highest cell death induction was observed in case of extracts obtained from herb of Th. vulgaris. Thymus vulgaris L. Concentration of Solanum tuberosum lectin was 0.4 mg/ml. The viability of J774 cells was assessed by propidium iodide (PI, 2 µg/ml) and Hoehst 33342 (4 µg/ml) staining using a fluorescence microscope OLYMPUS IX71S1F. PI-stained cells (red colour) were classified as necrotic, whereas PI-negative and weak Hoechst-positive (dim blue) cells were considered to be viable. * P<0.05 vs control. Fig. 2. Representative photographs of macrophage J774 cell culture in control and after treatment with lectin from Solanum tuberosum. Control Treatment