Fitohemijska i farmakološka karakterizacija odabranih biljnih vrsta roda Filipendula Mill. (Rosaceae)

Abstract

REZIME: Dve biljne vrste iz roda Filipendula Mill. (familija Rosaceae), Filipendula ulmaria (L.) Maxim. i Filipendula hexapetala Gilib., poznate kao medunika i suručica, su više- godišnje biljke koje se mogu naći na divljim i kultivisanim staništima u Evropi i Aziji. Obe biljke su korišćene u tradicionalnoj medicini mnogih evropskih zemalja, uključujući i Srbiju, zahvaljujući njihovim diuretičkim, antireumatskim i antiinfla- matornim osobinama. Međutim, naučnih saznanja o hemijskom sastavu F. ulmaria i F. hexapetala, kao i njihovoj biohemijskoj aktivnosti i farmakološkim efektima je vrlo malo. U tom smislu, glavni cilj ove doktorske disertacije bio je odrediti fitohemijski sadržaj i farmakološki potencijal ovih biljaka. U okviru ovih istraživanja kori- šćeni su metanolni ekstrakti nadzemnih delova i korena F. ulmaria (FUA i FUR) i F. hexapetala (FHA i FHR). Za određivanje hemijskog sastava ispitivanih ekstrakata primenjeno je nekoliko različitih tehnika, uključujući spektrofotometrijske metode za određivanje sadržaja jedinjenja fenolnog tipa, zajedno sa standardnim hromatografskim procedurama i hromatografskim procedurama visokih performansi (TLC, HPTLC, HPLC i LC-MS) za kvalitativnu i kvantitativnu analizu sadržaja fenola. Zadatak farmakološkog dela studije bio je istražiti antioksidativnu aktivnost ispitivanih metanolnih ekstra- kata, njihov potencijal u različitim model sistemima, kao i određivanje pH, termalne i gastrointestinalne stabilnosti. Zatim su primenjene metode za određivanje antimi- krobne aktivnosti na odabranim bakterijskim i fungalnim sojevima i citotoksično- sti na tri različite ćelijske linije. Takođe, u okviru ove disertacije ispitivan je antiinflamatorni efekat F. ulmaria and F. hexapetala u smislu njihovog uticaja na enzimsku aktivnost ciklooksigenaza (COX-1 i COX-2) i utvrđivanje antiinflamatornog potencijala, u in vitro i u in vivo uslovima. Štaviše, aktivnost ispitivanih ekstrakata F. ulmaria i F. hexapetala u borbi protiv stanja oksidativnog stresa izazvanog cisplatinom bila je ispitivana preko određivanja biohemijskih parametara seruma i tkiva (bubrega i jetre), kao i praćenjem histopatoloških promena i nivoa geno- i antigenotoksičnosti. Rezultati spektrofotometrijskih analiza F. ulmaria i F. hexapetala pokazali su znatnu količinu ukupnih fenolnih jedinjenja u ekstraktima, naročito flavonoida i fenolnih kiselina u nadzemnim delovima i kondenzovanih tanina i galotanina u ekstraktima korena. Prilikom TLC i HPTLC analiza FUA ekstrakt pokazao je prisustvo rutina, spireozida i izokvercitrina, a u FHA ekstraktu detektovani su kvercetin, spireozid, hiperozid i izokvercitrin. Primenom HPLC-PDA metode potvrđeno je prisustvo spireozida, kao referentne komponente, u ekstraktima nadzemnih delova obe biljne vrste. Prisustvo skoro svih standardnih jedinjenja bilo je ograničeno na nadzemne delove ove dve biljke, dok su u ekstraktima korena detektovani uglavnom samo katehin i epikatehin. Nakon hidrolize ekstrakata potvrđeno je da nadzemni delovi iv sadrže derivate kvercetina i kempferola koje nismo mogli detektovati HPLC anali- zom u nehidrolizovanim ekstraktima. Dalje, primenom LC-MS analize potvrđeno je da svi ispitivani ekstrakti poseduju različite klase fenolnih jedinjenja (oko 50 različitih jedinjenja), većinom fenolnih kiselina, flavonoida, tanina i njihovih derivata. Ekstrakti su ispoljili visoku aktioksidativnu aktivnost u poređenju sa referentnim antioksidantima, paralelno sa njihovim značajnim aktivnostima u procesu lipidne oksidacije i velikom stabilnošću u različitim uslovima. Ekstrakti su pokazali antimikrobne efekte na različite ispitivane mikroorganizme, posebno na bakterije E. coli i E. faecalis (MIC 0.156–0.625 mg/mL), kao i gljive P. cyclopium i F. oxysporum (MIC 2.5–5 mg/mL). F. ulmaria i F. hexapetala ekstrakti (FUA, FUR, FHA i FHR) nisu ispoljili in vitro citotoksičnu aktivnost u svim ispitivanim koncentracijama (IC50 > 50 μg/mL). Svi testirani ekstrakti u koncentraciji od 50 μg/mL inhibiraju enzimsku aktivnost COX-1 i -2, pri čemu su FUA i FHA ekstrakti bili skoro duplo efikasniji od ekstrakata korena odgovarajućih biljaka, sa izuzetkom inhibicije COX-2 od strane FHR ekstrakta (52.21%). Testirani materijali su vrlo slabo inhibirali ekspresiju COX-2 gena u THP-1 ćelijama, u koncentraciji od 25 μg/mL. U testu vruće ploče, kod pacova iz soja Wistar, svi ekstrakti, a posebno ekstrakti biljke F. hexapetala, u dve doze (100 i 200 mg/kg t.m.), izazvale su porast vremena latencije u poređenju sa kontrolnom grupom (p < 0.05). U testu akutne inflamacije indukovane karagenanom, ispitivani ekstrakti u dozama od 100 i 200 mg/kg t.m., delimično ili značajno redukuju maksimalni otok šape pacova sve do 6 h tretmana. Izloženost cisplatini u dozi od 7.5 mg/kg prouzrokovala je značajan porast biohemijskih parametara krvi koji ukazuju na promenu u normalnom funkcionisanju jetre i bubrega i markera oksidativnog stresa u tkivima, praćeno nekim histopatološkim promenama u tkivu bubrega i jetri eksperimentalnih životinja i visokim nivoom genotoksičnosti. Administracija ekstrakata F. ulmaria i F. hexapetala u tri različite koncentracije (100, 200 i 400 mg/kg/dan) u trajanju od 10 dana, rezultovala je smanjenjem oksidativnog stresa u tkivima i opadanjem serumskih parametara. Takođe, ispitivani ekstrakti smanjili su genotoksičnost cisplatine.

SUMMARY: Two plant species from genus Filipendula Mill. (fam. Rosaceae), namely Filipendula ulmaria (L.) Maxim. and Filipendula hexapetala Gilib., known as meadowsweet and dropwort, are perennial herbs found in wild and cultivated habitats in Europe and Asia. Both of them have been used in traditional medicine of most European countries including Serbia, based on theirs diuretic, antirheumatic and anti-inflammatory properties. However, there is little scientific evidence on chemical composition of F. ulmaria and F. hexapetala as well as their biochemical activity and pharmacological effects. In that regard, the main purpose of this PhD thesis was to evaluate the phytochemical composition and pharmacological potential of these plants. In this study were used methanolic extracts of aerial parts and roots of F. ulmaria (FUA and FUR) and F. hexapetala (FHA and FHR). For the phytochemical characterization several different techniques were applied, including spectrophotometrical methods for determination of the composition of different classes of phenolic compounds, together with standard and high performance chromatographical procedures (TLC, HPTLC, HPLC and LC-MS) for evaluation of phenolic composition qualitatively and quantitatively. The aim of pharmacological part of the study was to investigate the antioxidant activity of the tested methanolic extracts, their potential in different model systems, as well as pH, thermal and gastrointestinal stability studies. In addition, assays for determination of antimicrobial activity on selected bacterial and fungal strains and cytotoxicity on three different cell lines were carried out. Also, in this Dissertation were evaluated F. ulmaria and F. hexapetala anti-inflammatory effects regarding theirs impact on cyclooxygenases (COX-1 and COX-2) enzymatic activity and assessment of anti-inflammatory potential, both in in vitro and in vivo conditions. Moreover, activity of the tested extracts of F. ulmaria and F. hexapetala against cisplatin-induced oxidative stress in vivo was estimated via determination of serum and tissues (kidneys and liver) biochemical parameters, as well as monitoring histopathological changes and the levels of geno- and antigenotoxicity. The results of spectrophotometrical analysis of F. ulmaria and F. hexapetala showed considerable total content of phenolic compounds in the extracts, especially flavonoids and total phenolic acids in aerial parts and condensed tannins and gallotannins in root extracts. In TLC and HPTLC analysis FUA extract showed the presence of rutin, spiraeoside and isoquercitrin and in FHA extract quercetin, spiraeoside, hyperoside and isoquercitrin were detected. HPLCPDA analysis indicated presence of spiraeoside, as a reference compound, in the aerial part extracts of both plant species. The presence of almost all standard phenolic compounds was limited to the aerial parts of these two plants, whereas the root extracts contain only catechin and epicatechin. After hidrolysis of the extracts was confirmed that the aerial part extracts contain several derivatives of quercetin and kaempferol that we were unable detect via HPLC analysis before the hydrolysis. Further, in LC-MS analysis it was confirmed that all tested extracts possess a variety of phenolic compounds (around 50 different compounds), mainly phenolic acids, flavonoids, tannins and their derivatives. The extracts exerted high antioxidant vi activities compared to the reference antioxidants, paralleled by their considerable activities against lipid oxidation process and great stability in different conditions. The extracts revealed an interesting antimicrobial effect against the tested microorganisms, especially bacteria E. coli and E. faecalis (MIC 0.156–0.625 mg/mL), and fungi P. cyclopium and F. oxysporum (MIC 2.5–5 mg/mL). F. ulmaria and F. hexapetala extracts (FUA, FUR, FHA and FHR) had no in vitro cytotoxic activity at all applied concentrations (IC50 > 50 μg/mL). All tested extracts at a concentration of 50 μg/mL were able to inhibit COX-1 and -2 enzyme activities, whereby FUA and FHA extracts were double as effective as the root extracts of respective plants, with the exception of COX-2 inhibition by FHR extract (52.21%). Extracts hardly inhibited the level of COX-2 gene expression in THP-1 cells, at a concentration of 25 μg/mL. In the hot plate test using Wistar rats, all extracts, but especially extracts of F. hexapetala, in two doses (100 and 200 mg/kg b.w.), exhibited an increase in latency time when compared with the control group (p < 0.05). In the carrageenan-induced acute inflammation test, tested extracts at doses of 100 and 200 mg/kg b.w., were able to moderately or significantly reduce the mean maximal swelling of rat paw until 6 h of treatment. Exposure to cisplatin at a dose 7.5 mg/kg caused significant increase in blood biochemical parameters of liver and kidneys function and tissue oxidative stress markers along with some histopathological changes in kidney and liver tissues of experimental rats and high level of genotoxicity. Administration of F. ulmaria and F. hexapetala extracts at three different concentrations (100, 200 and 400 mg/kg/day) for 10 days resulted in a reduction of oxidative stress in tissues and decrease of serum parameters. Moreover, tested extracts attenuated the genotoxicity of cisplatin.