Chemical characterization and analysis of biological activity of isolates from birch (Betula pendula Roth.)

[M. S. Vladimirov]

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Biografija autora sa bibliografijom: list 150-154. Bibliografija: list. 128-147.

Chemistry and chemical technology

Research conducted within this doctoral dissertation includes the examination of the chemical composition, biological activities, as well as the potential for improving the properties of bioactive compounds derived from birch (Betula pendula Roth). For this purpose, different types of starting plant material were used, including birch buds, sap, leaves, and bark. In addition, inclusion complexes of standards of bioactive triterpenes from birch bark, betulin (BE) and betulinic acid (BA), with hydroxypropyl-β-cyclodextrin (HP-β-CD) were prepared, and their biological activity was also evaluated. Essential oils from birch buds and leaves were isolated by Clevenger-type hydrodistillation. Isolation of bioactive compounds from birch bark and leaves was performed by Soxhlet extraction, using 96% ethanol. The sap obtained from the birch tree was extracted by diethyl-ether and ethylacetate, after which the extracts were evaporated to dryness under reduced pressure using a rotary vacuum evaporator. The characterization of birch isolates was performed by gas chromatography–mass spectrometry (GC– MS). The bioactive triterpenes betulin and lupeol (LU) were identified in the birch bark extract. The content of micro- and macroelements in birch sap was determined using ICP-OES method. The mineral composition of birch sap indicated a relatively high content of the macroelements potassium (58.79 μg/cm³) and calcium (47.21 μg/cm³), while the most abundant microelement was manganese (25.72 μg/cm³). The total phenolic and flavonoid content in bark and leaf extracts, as well as in birch sap, were determined using the Folin-Ciocalteu method. It was found that the leaf extract contained the highest amount of total phenolics, whereas the lowest content of total phenolics was recorded in the sap extract. Total flavonoids were detected only in the birch leaf extract. Inclusion complexes of betulin and betulinic acid with hydroxypropyl-β- cyclodextrin were prepared by the coprecipitation method, in order to improve their water solubility. The complexes were characterized using various instrumental techniques (FTIR, SEM, XRD, DSC), which confirmed the successful inclusion of BE and BA into the cavities of cyclodextrin. Due to the increased water solubility, the BE:HP-β-CD inclusion complex exhibited stronger antioxidant activity compared to pure betulin. In contrast, betulinic acid showed lower antioxidant accitvity in the inclusion complex, which may be attributed to stronger interactions of its functional groups with HP-β-CD. The antimicrobial activity of essential oils was determined by the disk diffusion method, while the antimicrobial activity of extracts and inclusion complexes was detremined using the microdilution method. Birch leaf essential oil exhibited a broader spectrum of antimicrobial activity compared to birch bud essential oil. Birch bark extracts showed stronger antimicrobial activity compared to leaf extracts, while birch sap did not exhibit antimicrobial activity against the tested microorganisms. Betulin, betulinic acid, and their inclusion complexes did not show antibacterial activity against the tested bacterial strains. However, antimicrobial activity of betulinic acid and the BA:HP-β-CD complex was observed against the fungus Candida albicans, with more pronounced activity of betulinic acid in the inclusion complex. The antioxidant activity of isolates and formulations was evaluated by the DPPH assay. The obtained EC₅₀ values indicate that that the birch leaf extract exhibits the highest antioxidant activity, followed by the birch bark extract, while the lowest antioxidant activity was recorded in the birch sap extract. The effects of birch bark extract, birch sap, BE, BA, and the inclusion complexes of BE and BA on the viability and proliferation of MDCK cells in vitro were examined using the MTT assay. A stimulatory effect of the tested concentrations of birch sap on cell viability and proliferation was observed, whereas the birch bark extract exhibited cytotoxic and antiproliferative effects, at certain concentrations. Betulin and betulinic acid showed cytotoxic and anti-proliferative concentration-dependent effect, with a more pronounced effect observed for betulin. This trend was also observed in the case of inclusion complexes BE:HP-β-CD and BA:HP-β-CD, while the complexing agent (HP-β-CD) did not exhibit such effects at the tested concentrations.

breza, Betula pendula, betulin, betulinska kiselina, inkluzioni kompleksi antimikrobna aktivnost, antioksidativna aktivnost, MTT test, MDCK ćelijska linija

birch, Betula pendula, betulin, betulinic acid, inclusion complexes, antimicrobial activity, antioxidant activity, MTT test, MDCK cell line

615.322:582.631.1:543.5 (043.3)

T 410

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