Macedonian pharmaceutical bulletin, 68 (Suppl 2) 217 - 218 (2022) Online ISSN 1857 - 8969 DOI: 10.33320/maced.pharm.bull.2022.68.04.100 Short communication *k_danova@abv.bg S4 PP 03 Flavonoids productivity of wild growing and in vitro cultivated Hypricum species Antoaneta Trendafilova 1 , Viktoriya Ivanova 1 , Ina Aneva 2 , Kalina Danova *1 1 Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria 2 Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Introduction Over 1300 medicinal plants are being used in Europe, 90 % of them sourcing from Nature (Chen et al. 2016). Although widely studied throughout the years medicinal plant Hypericum perforatum still remains a source of scientific research due to the richness of its pharmacological activities, determined by the complexity of the phytochemical composition of Hyperici herba. Research has led to elucidation of the most important biologically active substances in the plant – polyphenolic compounds, flavonoids, naphthodianthrones and phloroglucinols, terpenes, possessing antidepressive, antitumor, antiviral and antibiotic activity (Danova 2015). The Balkan flora holds a priceless natural resource with a vast and still unexplored potential in the search of new sources of biologically active compounds. The aim of the present work was to compare the flavonoids productivity of three Hypericum species (H. perforatum, H. tetrapterum and H. richeri) collected from the wild with plant material derived from their biotechnological development. Materials and methods Plant material Hypericum species were collected from their natural habitats in Bulgaria as follows: H. perforatum and H. tetrapterum - Western Balkan region, H. richeri - from two different accessions - at the Vitosha and Rila mountains. Tissue culture initiation Shoot segments of the wild growing species (the samples of Vitosha Mountain for H. richeri) were surface sterilized by 30 sec. immersion in 70 % ethanol, followed by 5 min sterilization in 0.1 % HgCl2 and triplicate washing in sterile distilled water. For axillary shoot formation the following medium formulation was used: the basic Murashige and Skoog (MS) medium (1962), supplemented with Gamborg vitamins (Gamborg et al. 1968), 6.5 g/l agar, 20 g/l sucrose and 0.5 mg/l N 6 -benzyladenine (BA), at 25 ± 0.2 °C and 16/8 hours photoperiod. The obtained axillary shoots were transferred to the basic MS culture medium, supplemented with 30 g/l sucrose (with addition of 0.2 mg/l BA and 0.1 mg/l indole-3-butyric acid (IBA) for H. richeri). Shoot culture experiment The tissue culture experiment included the following plant growth regulator (PGR) treatments: Hyp_M0 - PGR- free control and Hyp_M2 - 0.2 mg/l BA + 0.1 mg/l IBA. Extraction of the plant material Dry plant material of the wild collected and shoot cultures derived Hypericum representatives (100 mg) was defatted by maceration in chloroform (3x10 ml) and further on extracted by methanol (3 x 10 ml). The extraction steps were 1 for 24 hr at room temperature, and 2 for 15 min in an ultrasonic bath at 25 °C. Total flavonoids determination Total flavonoids were assayed spectrophotometrically by the method of Zhishen et al. (1999) and the results were expressed as mg catechin equivalents per 1g DM.