Karakterizacija suvih ekstrakata cvasti smilja, Helichrysum plicatum DC. i ispitivanje njihove antioksidativne, citotoksične, spazmolitičke i antimikrobne aktivnosti / Characterization of dry extracts of everlasting inflorescences, Helichrysum plicatum DC

In this doctoral dissertation, the characterization of dry extracts of everlasting inflorescences, Helichrysum plicatum DC., and assay of their antioxidant, cytotoxic, spasmolytic and antimicrobial activity were carried out. Special attention was paid to investigation of correlation between chemical composition and pharmacological effects of dry extracts to determine potentially active substances or active markers. In the preformulation study, the herbal drug Helichrysi flos (Helichrysum plicatum DC.) was precisely defined, identified and tested. The dry extracts were prepared from powdered herbal drug by triple percolation method and purification of obtained liquid extracts by means of re-extraction method and vacuum evaporation. The following factors varied in the extraction procedures at three levels: degree of fineness of herbal drug, type of extragens (40%, 50%, 60% ethanol/water ratio) and re-extraction solvent (5:5, 9:1, 100:0 ethilacetate/ethanol ratio; v/v). The HPTLC fingerprint method was developed for rapid and reliable identification of glycosides and aglicone flavonoids present in the herbal drugs and extracts H.plicatum. This identification was based on the color fluorescence, and application of the standards. The tentative LC-UV-MS analysis of purified extracts revealed the presence of 18 compounds. Phenolic acids and flavonoids were also identified. As for flavonoids, there were four groups of compounds present: flavanones (naringenin and derivates), flavones (apigenin and derivates, luteolin), flavonols (quercetin and derivates; kaempeferol and derivates) and halcones (isosalipurpozide). The tentative LC-UV-MS analysis of drug and extract of everlasting inflorescences showed the difference between chromatograms of herbal drugs and extracts, because some compounds in herbal drug were present in small amounts and their presence was detected after extract purification process. A cynarin component was also identified that has not been identified in any of Helichrysum species. Thus obtained chemical profile H. plicatum DC. indicates a Summary Doctoral Dissertation significant similarity to H. arenarium (L.) Moench, and at the same time the differences present. In the chemical characterization of Helichrysi flos (Helichrysum plicatum DC.) dry extracts, the total content of phenols has been determined in all extract samples and it is in the range of 89,6 to 390,1 mg GAE/g. Statistically significant negative correlation has been established between extraction yields and total contents of phenols in extracts (r = - 0,74). Naringenin, apigenin and kaempeferol content was determined by HPLC methods in hydrolyzed and nonhydrolysed samples of dry extracts, and by application of standards. In all hydrolysed samples of the extracts, certain naringenin (248,6-754,7 mg/g) and apigenin content (19,7-138,5 mg/g) was identified and determined. Kaempeferol was identified in all samples, and quantified in several samples of extracts (4,5-193,9 mg/g). The results of naringenin (10,1-17,4 mg/g), apigenin (4,1-16,2 mg/g) and kaempeferol (0,7-3,8 mg/g) content determination in nonhydrolysed samples of dry extracts show that flavonoids in everlasting inflorescences are mainly in the form of glycosides. Extraction process optimization, based on factor analysis, showed different optimum extraction performance depending on the extraction target. In order to increase the dry extract yields, the extraction process optimization was based on the influence of various factors (degree of fineness of herbal drug, type of extragens and re-extraction solvent) on the extraction yields. The greatest effect of re-extraction solvent on extraction yields was observed as well as the degree of fineness of herbal drug - reextraction solvent interaction. For the purpose of increasing the total content of phenols, naringenin, apigenin and kaemferol, the extraction process optimization was based on the influence of variable factors (degree of fineness of herbal drug, type of extragens and re-extraction solvent) on total phenols, naringenin, apigenin and kaempeferol content. The higher dry extract yield was obtained by application of lower ethanol concentration in extragens (50%) and ethyl acetate : ethanol 5:5 (re-extraction solvent), while the higher content of phenols and flavonoids was obtained by application of higher ethanol concentration in extragens (60%) and ethyl acetate : ethanol 9:1 (reextraction solvent). The optimum degree of fineness of herbal drug used for extraction Summary Doctoral Dissertation contained 53-57% of fraction in the range of 355-2000 µm and 42-45% of fraction >2000 µm. Antioxidant activity of dry extract samples was evaluated on the basis of the ability of DPPH radical reduction. The extracts of Helichrysi flos (Helichrysum plicatum DC.) showed different antioxidant activities (IC50 values 37,17-88,61 µg/mL), lower than the activity of reference substance. The extracts rich in total phenols showed the highest antioxidant potential. Cytotoxic activity of selected extracts of Helichrysi flos (Helichrysum plicatum DC.) and standard substances (naringenin, apigenin and kaempeferol) was tested in vitro on three types of human cancer cells: Cervix Aden carcinoma Hela cells, PC3 prostate cancer cell lines and myelogenous leukemia K562 cells. All tested extracts exhibited moderate activity against Hela cells (IC50 value 41,9-42,1 µg/ml), whereas the extract containing the highest total phenols was the most active against K562 cells ( IC50 value 25,9 µg/ml) and PC3 lines (IC50 value 39,2 µg/ml). Among aglycones tested, kaempeferol displayed strong cytostatic activity against all cell lines. The results of in vitro spasmolytic activity testing of selected extract of Helichrysi flos (Helichrysum plicatum DC.), assayed on isolated rat ileum, showed spasmolytic effect of dry extract on spontaneous ileum contractions and contractions induced by acetylcholine, histamine, barium and potassium ions. Cumulative concentrations of the extract induced a spasmolytic effect on spontaneous rat ileum contractions and caused a mean contractile response of 81,68 % (at the concentration of 0,01 mg/ml) and 30,08 % (at the concentration of 1 mg/ml). A similar effect was observed with papaverine (0,01–3 μg/ml) that was used as the reference substance. The extract (0,01–1 mg/ml) relaxed high K + (80 mM) precontractions, an effect similar to that caused by papaverine (0,01–3μg/ml). The plant extract (0,03–0,3 mg/ml) also induced a significant depression of the cumulative concentration response curve for acetylcholine (5–1500 nM) (p < 0,01). Atropine (140 nM) abolished the acetylcholine effect. The extract (0.03–0.3 mg/ml) reduced the contractions induced by the histamine (1–300 nM) and BaCl2 (3–900 μM) (p < 0,01). Antimicrobial activity of selected extract of Helichrysi flos (Helichrysum plicatum DC.) was tested by in vitro micro dilution method against 20 microorganisms. The results indicated its strong antibacterial (MIC values 0,010 – 0,055 mg/ml), and antifungal potential (MIC values of 0,005 - 0,04 mg/ml). The most sensitive was B. subtilis (MIC value of 0,01 mg/ml), but the most resistant was E. coli (MIC value of 0,055 mg/ml). The extract inhibited the growth of yeast C. albicans (MIC value 0,02 mg/ml). Phytopathogene yeasts were more sensitive than bacteria on the tested extract (MIC values of 0,005-0,04 mg/ml). Potential aflatoxin producer, A. flavus, was inhibited at very low concentrations (MIC 0,04 mg/ml). These results enable the application of the extract to the protection of medicinal plants against plant pathogenic fungi. There is also a possibility of everlasting extract use in the food industry as a natural preservative. The results of Helichrysi flos (Helichrysum plicatum DC.) chemical composition and pharmacological activity analyses indicated the following: a) positive correlation between antioxidant activity extracts and total phenols content (r = 0,79), b) positive correlation between antioxidant activity extracts and kaempeferol content (r = 0.83), c) positive correlation between extract cytotoxic activity against PC3 and K562 cells and total phenols content (r = 0,98 and r = 0,99), d) positive correlation between extract cytotoxic activity against PC3 and K562 cells and kaempeferol content (r = 0,88 and r = 0,94), e) positive correlation between extract cytotoxic activity against PC3 and K562 cells and their antioxidant activity (r = 0,94 and r = 0,98). These correlations suggest that the total phenols / kaempeferol could be active substances or active markers used for H.flos extract standardization (biological source Helichrysum plicatum DC.) when the antioxidant/cytotoxic effect is expected. The samples of extracts, that showed spasmolytic and strong antimicrobial activity, had high total phenol content and all determined aglycones of naringenin, apigenin and kaempeferol, and these results suggest a correlation between the content of total phenols and flavonoids with spasmolytic, antibacterial and antifungal activities. The results, as demonstrated above, justify the traditional use of H. plicatum DC. and indicate the similarity to H. Arenarium (L.) Moench, which is a good basis for further investigations.