Fitohemijska i farmakološka karakterizacija odabranih biljnih vrsta roda Filipendula Mill. (Rosaceae)
Phytochemical and pharmacological characterization of selected plant species of the genus Filipendula Mill. (Rosaceae)
Author
Katanić, Jelena
Mentor
Mihailović, Vladimir
Committee members
Mišić, DanijelaBauer, Rudolf
Rosić, Gvozden
Mladenović, Milan
Metadata
Show full item recordAbstract
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 spektr...ofotometrijske 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.