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Predmet doktorske disertacije je analiza hemijskog sastava i ispitivanje farmakološke aktivnosti izolata (etarskih ulja, hloroformskog i metanolnog ekstrakta i metabolita) podzemnih organa srpske velestike, Ferula heuffelii Griseb. ex Heuffel (Apiaceae). Srpska velestika je endemična vrsta koja naseljava klisure i stenovite terene istočne Srbije, jugozapadne Rumunije i zapadne Bugarske. Ova vrsta do sada nije ispitivana u pogledu hemijskog sastava i farmakološke aktivnosti. Etarska ulja podzemnih organa F. heuffelii izolovana su iz uzoraka prikupljenih na tri lokaliteta u Republici Srbiji: Sićevačkoj klisuri, Đerdapskoj klisuri i klisuri reke Pek. Etarska ulja su izolovana destilacijom vodenom parom i hemijski okarakterisana metodama GC-FID i GC-MS. Etarsko ulje izolovano iz podzemnih organa poreklom iz Sićevačke klisure (F1) bilo je bogato seskviterpenima (48,7%) i fenilpropanima (17,0%), a glavni sastojak bio je elemicin (12,5%). Etarska ulja izolovana iz podzemnih organa prikupljenih u Đerdapskoj klisuri (F2) i klisuri reke Pek (F3), karakterisao je visok sadržaj fenilpropanskih jedinjenja (56,0 i 29,7%, redom), pri čemu su elemicin (35,4 i 16,8%, redom) i miristicin (20,6 i 12,9%, redom), bili najzastupljenije komponente. Razlike između ispitivanih etarskih ulja bile su pre svega kvantitativne prirode (...), The scope of this doctoral dissertation represents chemical analysis of underground parts of Ferula heuffelii Griseb. ex Heuffel (Apiaceae), and investigation of pharmacological activity of its isolates (chloroform and methanol extracts, essential oils and metabolites). Ferula heuffelii („srpska velestika“) is an endemic and rare perennial species which grows predominantly in gorges and stony places in Eastern Serbia, and locally in Southwestern Romania and Western Bulgaria. This plant has not been previously investigated for chemical composition and pharmacological activity. The essential oils of underground parts of F. heuffelii were isolated from the samples collected from three localities in Serbia: the Sićevo Gorge; the Djerdap Gorge and the gorge of the river Pek. The oils were isolated by hydrodistillation in a Clevenger-type apparatus and analyzed by GC-FID and GC-MS methods. The oil from underground parts obtained from the Sićevo gorge (F1) was rich in sesquiterpene hydrocarbons (48.7%) and phenylpropanoids (17%), and elemicin was the major constituent (12.5%). The oils isolated from underground parts collected in the Djerdap gorge (F2) and in the gorge of the river Pek (F3), were characterized by high content of phenylpropanoids (56.0 and 29.7%, respectively), with elemicin (35.4 and 16,8%, respectively) and myristicin (20.6 and 12,9%, respectively) being the main compounds. The differences in chemical composition between the essential oils were mainly quantitative (...)

Antimicrobial properties of extracts of underground parts of three Laserpitium L. (Apiaceae) species, namely Laserpitium latifolium L., Laserpitium zernyi Hayek and Laserpitium ochridanum Micevski, were investigated. The investigated species are widely used as functional foods, as spices and for preparations in traditional medicine for treating complaints connected with infection and inflammation. Furthermore, antimicrobial and antibiofilm effects of laserpitine, the most abundant compound in the chloroform extract of Laserpitium latifolium, and guaianolide sesquiterpene lactones, such as, isomontanolide, montanolide and tarolide, principal components of the extracts of Laserpitium zernyi and Laserpitium ochridanum were assessed. The antimicrobial activity was tested using the microdilution method against five pathogenic bacteria and five fungi, as well as in the microplate biofilm assay on two Candida clinical isolates (C. albicans and C. krusei). Among the extracts, Laserpitium latifolium showed the most prominent activity. Isolated metabolites exerted higher effects against fungal than against bacterial strains, isomontanolide being the most active. Interestingly, all constituents showed higher potential on inhibition of biofilm formation than fluconazole, a reference compound. Tested metabolites may be good novel agents with high antifungal and antibacterial potential that might find practical applications in food industry as food preservatives in order to retard the growth of food spoiling microbes, but only after detailed safety assessments.

The chemical composition and antimicrobial activity of essential oils of Laserpitium latifolium and L. ochridanum were investigated. The essential oils were isolated by steam distillation and characterized by GC-FID and GC/MS analyses. All essential oils were distinguished by high contents of monoterpenes, and alpha-pinene was the most abundant compound in the essential oils of L. latifolium underground parts and fruits (contents of 44.4 and 44.0%, resp.). The fruit essential oil was also rich in sabinene (26.8%). Regarding the L. ochridanum essential oils, the main constituents were limonene in the fruit oil (57.7%) and sabinene in the herb oil (25.9%). The antimicrobial activity of these essential oils as well as that of L. ochridanum underground parts, whose composition was reported previously, was tested by the broth-microdilution method against four Gram-positive and three Gram-negative bacteria and two Candida albicans strains. Except the L. latifolium underground-parts essential oil, the other investigated oils showed a high antimicrobial potential against Staphylococcus aureus, S. epidermidis, Micrococcus luteus, or Candida albicans (minimal inhibitory concentrations of 13.0-73.0 mu g/ml), comparable to or even higher than that of thymol, which was used as reference compound.

The antimicrobial and cytotoxic activities of isolates (CHCl3 and MeOH extracts and selected metabolites) obtained from the underground parts of the Balkan endemic plant Ferula heuffeliiGriseb. ex Heuff. were assessed. The CHCl3 and MeOH extracts exhibited moderate antimicrobial activity, being more pronounced against Gram-positive than Gram-negative bacteria, especially against Staphylococcus aureus (MIC=12.5g/ml for both extracts) and Micrococcus luteus (MIC=50 and 12.5g/ml, resp.). Among the tested metabolites, (6E)-1-(2,4-dihydroxyphenyl)-3,7,11-trimethyl-3-vinyldodeca-6,10-dien-1-one (2) and (2S*,3R*)-2-[(3E)-4,8-dimethylnona-3,7-dien-1-yl]-2,3-dihydro-7-hydroxy-2,3-dimethylfuro[3,2-c]coumarin (4) demonstrated the best antimicrobial activity. Compounds 2 and 4 both strongly inhibited the growth of M. luteus (MIC=11.2 and 5.2M, resp.) and Staphylococcus epidermidis (MIC=22.5 and 10.5M, resp.) and compound 2 additionally also the growth of Bacillus subtilis (MIC=11.2M). The cytotoxic activity of the isolates was tested against three human cancer cell lines, viz., cervical adenocarcinoma (HeLa), chronic myelogenous leukemia (K562), and breast cancer (MCF-7) cells. The CHCl3 extract exhibited strong cytotoxic activity against all cell lines (IC50 lt 11.0g/ml). All compounds strongly inhibited the growth of the K562 and HeLa cell lines. Compound 4 exhibited also a strong activity against the MCF-7 cell line, comparable to that of cisplatin (IC50=22.32 +/- 1.32 vs. 18.67 +/- 0.75M).

In this paper antinociceptive and anti-edematous effects are examined of the essential oils of the underground parts of two Balkan endemic Laserpitium species (Apiaceae), L. zernyi and L. ochridanum. Furthermore, the essential oil of the underground parts of L. ochridanum is chemically characterised by GC and GC-MS. Antinociceptive and anti-edematous effects were measured in a rat model of localized inflammation, induced by carrageenan, using apparatus for the modified paw-pressure test, and plethysmometer, respectively. The effects of both Laserpitium essential oils were measured after oral gavage administration to male Wistar rats in doses of 25, 50 and 100 mg/kg. The main constituents of L. ochridanum essential oil were: alpha-pinene (33.2%), alpha-bisabolol (10.3%) and chamazulene (14.9%). The essential oil of L. zernyi was previously shown to be rich in alpha-pinene (31.6%) and alpha-bisabolol (30.9%). Both examined essential oils produced a significant dose-dependent antinociceptive effect. The corresponding ED50 +/- SEM in producing antinociception were 45.9 +/- 4.9 mg/kg and 42.4 +/- 2.1 mg/kg for L. zernyi and L. ochridanum oil, respectively. Both essential oils also significantly reduced paw edema in a dose-dependent manner. The estimated ED50 +/- SEM values for the anti-edematous effect were 36.3 +/- 4.5 mg/kg for L. zernyi oil and 45.1 +/- 11.3 mg/kg for L. ochridanum oil. These results suggest that the essential oils of both investigated Laserpitium species may be effective against pain and edema present in various inflammatory conditions.

Plants from the genus Ferula L. (Apiaceae) were used for various purposes in traditional medicine of different nations throughout the history. Ferula heuffelii GRISEB. ex HEUFFEL is a perennial species endemic for Balkan peninsula. Ten compounds which belong to classes of prenyl-furocoumarin-, prenyl-dihydrofurochromone-, prenyl-benzoyl-and prenyl-benzoylfuranone-type sesquiterpenoids, as well as sesquiterpene coumarins and phenylpropanoids, were, for the first time, isolated from the CHCl3 extract of the underground parts of this plant and identified. Furthermore, extract and three isolated compounds, i.e., latifolone (1), dshamirone (4), and (2S*, 3R*)-2-[(3E)-4,8-dimethylnona-3,7-dien-1-yl]-2,3-dihydro7- hydroxy-2,3-dimethylfuro[3,2-c] coumarin (6) were, for the first time, evaluated for their in vitro antispasmodic activities in three experimental models: spontaneous contraction, and ACh- and KCl-induced contraction of an isolated rat ileum. The extract (0.1-1.3 mg/ml) and compound 6 (1 - 10 mu g/ml) exhibited dose-dependent effect in all three models. Compound 1 (1-6 mu g/ml) affected spontaneous contractions and those induced by KCl, while compound 4 (8 mu g/ml) displayed only moderate activity with ACh-induced contractions. It can be concluded that tested compounds contribute to exhibited antispasmodic activity of crude extract. Additionally, extract (0.1-1.3 mg/ml) was tested for in vitro relaxant activity on an isolated rat trachea, and relaxed the KCl-induced contractions in a dose-dependent manner.

In the search for novel cytotoxic agents, sesquiterpenoids and phenylpropanoids have provided interesting lead compounds. From the chloroform extract of the underground parts of Laserpitium latifolium, the daucane sesquiterpenoids laserpitin and acetyldesoxodehydrolaserpitin, and the phenylpropanoids laserin and latifolon were isolated as the major compounds. Acetyldesoxodehydrolaserpitin is identified for the first time in the genus Laserpitium. Using a MTT and a sulforhodamine B (SRB) assay, the cytotoxic and antiproliferative activity of the extract and compounds laserpitin, acetyldesoxodehydrolaserpitin and laserin was tested in two closely related human breast adenocarcinoma cell lines, ie. the invasive MCF 7/6 and the non-invasive MCF 7/AZ. The IC50 values of the extract were in the range of 184.72 - 397.16 mu g/mL, with the most potent effect observed in the MTT test on the MCF 7/6 line. Among the tested compounds, acetyldesoxodehydrolaserpitin exerted a most potent, concentration-dependent effect (IC50 values of 0.60 and 0.51 mu M in the MCF 7/6 cell line, and 2.29 mu M and 31.87 mu M in the MCF 7/AZ cell line in the MTT and SRB test, respectively). The effect of laserin was more pronounced in the MTT test (IC50 of 4.57 and 2.46 mu M in the MCF 7/6 and MCF 7/AZ, respectively).

The essential oil from underground parts of Ferula heuffelii from N.E. Serbia, was analysed using GC and GC-MS. The main compounds of the essential oil were elemicin (35.4%) and myristicin (20.6%). The essential oil exhibited the best antimicrobial activity against two strains of Candida albicans (MIC = 7.0 and 13.7 mu g/ml), as well as against Micrococcus luteus (MIC = 13.7 mu g/ml), Staphylococcus epidermidis (MIC = 17.6 mu g/ml), Bacillus subtilis (MIC = 21.1 mu g/ml) and Micrococcus flavus (MIC = 28.2 mu g/ml). In the DPPH radical scavenging assay, essential oil showed substantial activity with SC(50) = 22.43 mu l/ml. The essential oil was also tested for antispasmodic activity. It inhibited spontaneous contraction of isolated rat ileum dose-dependently, and at the concentration of 86.64 mu g/ml exhibited 50% of the maximum effect of atropine. After incubation with 75.00 mu g/ml of essential oil, acetylcholine did not induce contractions of ileum, and at 250.00 mu g/ml, the essential oil almost completely abolished the spasmodic effect of potassium chloride (80 mM).

The present study describes investigation on chemical profile and pharmacological properties of hydromethanolic extracts of Carlina acaulis and C. acanthifolia roots and herbs. In order to investigate the antioxidant properties several methods were applied: FRAP (Ferric Reducing Antioxidant Power) assay, TBA assay (Fe2+/ascorbic induced lipid peroxidation) and scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and OH radicals. In assessing anti-inflammatory activity the carrageenan-induced rat paw oedema test was used, with indomethacin as a reference drug. Anti-ulcer activity of the extracts was investigated using ethanol-induced stress ulcer in rats and ranitidine as a reference drug. Antimicrobial activity was tested on five bacterial strains and one strain of fungi using the agar diffusion method. Investigated extracts exhibited considerable dose-dependent antioxidant, anti-inflammatory and anti-ulcer activities. Extracts of herbs were more active than the corresponding root extracts. All extracts exhibited moderate activity against all tested microorganisms. Herb extracts of C. acaulis and C. acanthifolia, as well as their root extracts exhibited similar bioactivity, which could be explained with their similar chemical composition revealed by HPLC analysis. The pharmacological activities of investigated Carlina extracts demonstrated here will serve as the basis for their further investigations in order to estimate their impact on human health and nutrition.

The essential oil obtained by hydrodistillation from the roots of Anthriscus nemorosa (Bieb.) Sprengel (Umbelliferae) was analyzed by GC and GC-MS. Among sixty-two compounds identified (representing 89.0% of the total oil), the main components were: n-nonane (12.1%), n-hexadecanol (6.9%), delta-cadinene (6.4%), beta-pinene (6.0%) and germacrene D (5.4%). Furthermore, the antimicrobial activity of the oil was evaluated against the Gram-positive bacteria Staphylococcus epidermidis (ATCC 12228) and Bacillus subtilis (ATCC 6633), the Gram-negative bacterium Escherichia coli (ATCC 25922), and a yeast Candida albicans (ATCC 10259 and ATCC 24433) using the broth microdilution method.

The chemical composition and free radical scavenging properties of the MeOH extract of Hypericum richeri and its subsequent CHCl3, n-BuOH, EtOAc and aqueous extracts were investigated. Four quercetin-3-O-glycosides, three myricetin-3-O-glycosides, 3-O- and 5-O-caffeoylquinic acid, quercetin, I3,II8-biapigenin, pseudohypericin and hypericin were identified. The constituents of the extracts were quantified using HPLC-DAD (high-performance liquid chromatography-diode array detector). Isolation of the compounds from n-BuOH and EtOAc extracts was conducted by column chromatography. Antioxidant capacity of the extracts was evaluated by superoxide radical and ABTS root+ scavenging assays and inhibition of lipid peroxidation. The EtOAc extract of H. richeri was the most potent antioxidant in all experiments. Free radical scavenging compounds were identified by HPLC-DPPH root (high-performance liquid chromatography-2,2-diphenyl-1-picrylhydrazyl) post-column derivatisation method, where all phenolic compounds except I3,II8-biapigenin exhibited radical scavenging properties. This is the first report of the presence of myricetin-3-O-rutinoside, myricetin-3-O-galactoside, myricetin-3-O-glucoside, 5-O-caffeoylquinic acid and I3,II8-biapigenin in H. richeri.

The influence of methanol extracts of four Stachys species from Balkan on carbon tetrachloride induced hepatotoxicity in rats was investigated. The extracts were obtained from aerial parts of S. beckeana Dorfler & Hayek, S. anisochila Vis. et Pancic, S. plumosa Griseb. and S. alpina L. subsp. dinarica Murb. The liver damage was induced by s.c. injections of carbon tetrachloride (2.5 ml/kg b.w.) and the extracts were then consecutively applied for five days. Their effects were evaluated through alteration of biochemical parameters (liver enzymes in the serum), as well as through histopathological changes in the liver (liver damage score, LDS). Treatment of CCl(4)-intoxicated rats with methanol extracts of investigated Stachys taxa (200 and 100 mg/kg b.w. p.o.) significantly reduced increased level of marker enzymes aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) in the serum (with the exception of AST and ALT after 100 mg/kg of S. anisochila extract was administered). Vast degenerative and vascular changes in CCl(4)-treated rats were also notably reduced after the treatment with investigated extracts, corroborating the biochemical observations and confirming their hepatoprotective effects. The best dose-dependant activity was achieved by the methanol extract of S. alpina subsp. dinarica.

Oil extracts of flowering tops of Hypericum richeri Vill. prepared in three different ways were evaluated for chemical composition, and anti-inflammatory and gastroprotective activities. An HPLC method was developed for determination of two dominant flavonoids, quercetin and I3,II8-biapigenin. The carrageenan-induced rat paw edema test was used for screening the anti-inflammatory activity, while indomethacin-induced rat gastric mucosa damage test was used for evaluation of gastroprotective activity. The oil extract prepared by maceration with 96% ethanol, followed by extraction with sunflower oil by heating on a water bath, exhibited the highest anti-inflammatory (38.4 %) and gastroprotective activities (gastric damage score of 0.9). The same oil extract had the highest content of quercetin (49 mu g/mL) and I3,II8-biapigenin (60 mu g/mL). These results approve the usage of oil extracts of H. richeri as an anti-inflammatory and gastroprotective agent.

Oil extracts of Hypericum perforatum L. (Oleum Hyperici) were prepared in three different ways according to the prescriptions from traditional medicine. Variability of constituents and biological activity were evaluated in the obtained oil extracts. The carrageenan-induced rat paw edema test has been used for screening the antiinflammatory activity, while the indomethacin-induced rat gastric mucosa damage test was used for evaluation of gastroprotective activity. All examined oil extracts possessed antiinflammatory and gastroprotective activity. Among them, the oil extract prepared by maceration with 96% ethanol, followed by extraction with sunflower oil by heating on a water bath (extract 2), in a dose of 1.25 mL/kg p.o., exhibited the highest antiinflammatory effect (95.24 +/- 11.66%) and gastroprotective activity (gastric damage score of 0.21 +/- 0.12). The same oil extract had the highest content of quercetin and I3,II8-biapigenin (1.29 +/- 9 mu g/mL and 52 +/- 4 mu g/mL, respectively). Quercetin and I3,II8-biapigenin exhibited antiinflammatory activity similar to those of indomethacin as well as significant gastroprotective activity. The results provide evidence for the usage of Oleum Hyperici as an antiinflammatory and gastroprotective agent, Which has been based previously only on ethnopharmacological claims. Copyright

The results of the chemical investigations of the essential oils from the flowers, leaves, sterns and rhizome of Peucedanum officinale are presented. The essential oil contents were 1.21% (v/w) in flowers, 0.64% (v/w) in leaves, 0.08% (v/w) in stems and 0.24% (v/w) in rhizome. By CC and GC/MS analysis, in all four oils the total of 132 compounds; was identified: 53 constituents (93.4% of total amount) in the oil obtained from the flowers, 70 (94.3% of total amount) in the oil from the leaves, 78 (87.8% of total amount) in the oil from the sterns and 67 (95.9% of total amount) in the oil from rhizome. in all oils investigated, monoterpenes were the dominant class of constituents (73.9-83.7%), with hydrocarbons (65.6-81.6%) as the most abundant representatives. Limonene, alpha-pinene and sabinene were identified as the most important constituents in common for all flour oils. In the oil from the flowers, leaves and sterns, beta-pinene and myrcene were also abundant. However, the flower oil differed from the others by a significant quantity of alpha-phellandrene.