Vrste roda Hieracium s. str. sa Balkanskog poluostrva: sekundarni metaboliti i farmakološki potencijal

The hawkweed genus Hieracium L. s. str. (Asteraceae) is one of the species-richest and systematically the most complex genera of flowering plants. The genus traditional classification, beside Hieracium, included, among others, subgenus Pilosella, which is currently considered as separate genus. In folk medicine, application of some Hieracium s. str. species was reported for various respiratory, gastrointestinal, urogenital, and some skin disorders. The 28 species from the Balkan Peninsula were investigated, and majority was collected on the Mt Durmitor (Montenegro), where the greatest diversity of taxa of this genus was registered. In flowering aerial parts, composition of pharmaceutically significant secondary metabolites - flavonoids, phenolic acids, sesquiterpene lactones and triterpenes was established, and by multivariate statistical analysis their chemosystematic significance was demonstrated. Corresponding metabolites isolation was performed using column chromatography and semi-preparative liquid chromatography (LC-MS), identification using mass spectrometry, NMR spectroscopy and refractometry, and extracts comparative qualitative and quantitative analysis using LC-MS or gas chromatography (GC-FID-MS). In all 28 species, sesquiterpene lactones were identified for the first time. Three of them were previously undescribed proline conjugates. Sesquiterpene lactone-amino acid conjugates are quite rare in nature, and formerly were identified only in a few species of other Asteraceae genera. Results obtained by investigation of pharmacological activities (in vitro antioxidant, antimicrobial, cytotoxic, anticholinesterase, and in vivo antihyperalgesic and antiedematous), as well as safety profiles of selected, chemically characterized extracts and/or secondary metabolites, indicated the medicinal potential of the analysed plants and gave justification for further research aiming to find new natural medicinal raw materials.Rod Hieracium L. s. str. (jastrebnik, runjavica) (Asteraceae) je jedan od najvećih i sistematski najsloženijih rodova skrivenosemenica. Tradicionalna klasifikacija roda je pored podroda Hieracium uključivala, između ostalog, i podrod Pilosella, koji prema savremenoj klasifikaciji ima status roda. Za pojedine vrste roda Hieracium s. str. je zabeležena primena u narodnoj medicini kod različitih poremećaja respiratornog, gastrointestinalnog, urogenitalnog trakta i kod nekih kožnih bolesti. Sprovedeno je istraživanje 28 vrsta sa Balkanskog poluostrva, od kojih je najveć i broj sakupljen na planini Durmitor (Crna Gora), na kojoj je konstatovan najveći diverzitet taksona roda. U nadzemnim delovima biljaka u cvetu ustanovljena je kompozicija sekundarnih metabolita od značaja za farmaciju - flavonoida, fenolkarboksilnih kiselina, seskviterpenskih laktona i triterpena, i multivarijantnom statističkom analizom pokazan i njihov hemosistematski značaj. Izolovanje odgovarajućih metabolita izvršeno je primenom hromatografije u koloni i semi-preparativne tečne hromatografije (LC-MS), identifikacija primenom masene spektrometrije, NMR spektroskopije i refraktometrije, a uporedna kvalitativna i kvantitativna analiza ekstrakata primenom LC-MS ili gasne hromatografije (GC-FID- MS). Seskviterpenski laktoni su u svih 28 vrsta identifikovani po prvi put, od kojih su tri do sada neopisani konjugati sa prolinom. Konjugati seskviterpenskih laktona sa aminokiselinama su inače vrlo retki u prirodi i do sada su identifikovani samo u nekoliko vrsta drugih rodova familije Asteraceae. Rezultati dobijeni ispitivanjem farmakološke aktivnosti (in vitro antioksidantne, antimikrobne, citotoksične, antiholinesterazne, i in vivo antihiperalgezijske i antiedematozne), kao i rezultati ispitivanja bezbednosti primene za odabrane, hemijski okarakterisane ekstrakte i/ili sekundarne metabolite ukazali su na lekoviti potencijal ispitivanih biljaka i opravdanost daljih istraživanja u cilju iznalaženja novih prirodnih lekovitih sirovina.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Investigation of secondary metabolites and pharmacological activity of selected species of the genus Hieracium L. (Asteraceae) from the Balkan Peninsula

hibridogenih). Semipreparativnim LC-MS metodama, iz MeOH ekstrakta cvasti H. calophyllum izolovana su četiri seskviterpenska laktona (krepizid E i tri nova jedinjenja: 8-epi-ikserisamin A, kalofilamin A i kalofilamin B), a iz MeOH ekstrakta podzemnih organa H. scheppigianum pet fenolkarboksilnih (mono- ili di-O-kafeoilhina) kiselina (identifikacija na osnovu NMR i MS spektara). Ukupno četiri seskviterpenska laktona, sedam mono- ili di-O-kafeoilhina kiselina i 19 flavonoida (luteolin, apigenin, diosmetin, 15 njihovih heterozida i heterozid kvercetina) kvantifikovani su u suvim MeOH ekstraktima ispitivanih podzemnih organa dve vrste i herbi 28 vrsta LC-MS metodama, i pet triterpena (derivata ursana, oleana i lupana) u suvim CH2Cl2 ekstraktima herbi 28 vrsta GC-FID-MS metodom (identifikacija pomoću standarda, NMR, MS i/ili UV spektara). Hemosistematski značaj jedinjenja identifikovanih u herbama 28 vrsta utvrđen je multivarijantnim statističkim metodama (PCA, nMDS i UPGMA). Suvi MeOH ekstrakti herbi su in vitro pokazali značajan ukupni antioksidantni potencijal i sposobnost neutralizacije slobodnih DPPH•, ABTS•+ i OH• radikala (28 vrsta) i inhibicije acetilholinesteraze (27 vrsta) i butirilholinesteraze (7 vrsta). Među testiranih 12 jedinjenja, najbolju antiholinesteraznu aktivnost ispoljila su tri flavonoidna aglikona i 8-epi-ikserisamin A. Odabrani suvi MeOH i CH2Cl2 ekstrakti herbi šest vrsta pokazali su antimikrobnu aktivnost prema 18, a 8- epi-ikserisamin A i krepizid E prema 4 mikroorganizma. Isti odabrani izolati ispoljili su citotoksični efekat prema tri, odnosno jednoj tumorskoj ćelijskoj liniji, uz zanemarljivu inhibiciju rasta zdravih ćelija. Tri odabrana MeOH i CH2Cl2 ekstrakta su u modelu inflamacije šape pacova izazvane karageninom pokazala antihiperalgezijsku, ali ne i antiedematoznu aktivnost; bezbednost primene dva ekstrakta koji su ispoljili značajnu antihiperalgezijsku aktivnost pokazana je in vivo rotarod i testom akutne toksičnosti. Ključne reči:nonhybridogenous and 16 hybridogenous). Using semipreparative LC-MS methods, four sesquiterpene lactones (crepiside E and three new compounds: 8-epiixerisamine A, calophyllamine A and calophyllamine B) were isolated from the MeOH extract of H. calophyllum flowering heads, and five phenolic (mono- or di-Ocaffeoylquinic) acids from the MeOH extract of H. scheppigianum underground parts (identification based on NMR and MS spectra). Total of four sesquiterpene lactones, seven mono- or di-O-caffeoylquinic acids and 19 flavonoids (luteolin, apigenin, diosmetin, their 15 glycosides and one quercetin glycoside) were quantified in the dried MeOH extracts of the investigated underground parts of two species and herbs of 28 species, using LC-MS methods, and five triterpenes (ursane, oleane and lupane derivatives) in the dried CH2Cl2 extracts of the herbs of 28 species, using GC-FID-MS method (identification using standards, NMR, MS and/or UV spectra). Chemosystematic significance of the compounds identified in the herbs of 28 species was determined using multivariate statistics (PCA, nMDS and UPGMA). Dried MeOH herb extracts in vitro exhibited significant total antioxidant activity and scavenging of DPPH•, ABTS•+ and OH• radicals (28 species), as well as the ability to inhibit acetylcholinesterase (27 species) and butyrylcholinesterase (7 species). Among 12 tested compounds, three flavonoid aglycones and 8-epiixerisamine A exhibited the highest anticholinesterase activity. Selected dried MeOH and CH2Cl2 herb extracts of six species showed antimicrobial activity against 18, whereas 8-epiixerisamine A and crepiside E against four microorganisms. The same selected isolates exhibited cytotoxic activity against three and one cancer cell lines, respectively, with negligible inhibition of the normal cells growth. In the carrageenan-induced localized inflammation model in rats, three selected MeOH and CH2Cl2 extracts exhibited antihyperalgesic, but not antiedematous activity; safety of the oral administration of two extracts that exhibited significant antihyperalgesic activity was demonstrated in vivo in rotarod test and acute oral toxicity study

Sekundarni metaboliti i antiholinesterazna aktivnost herbe Chamaecytisus heuffelii subsp. jankae

Chamaecytisus heuffelii subsp. jankae (Velen.) Niketić (Fabaceae) is a dwarfish shrub, which range includes eastern part of the Balkan Peninsula (Albania, Bulgaria, Serbia) (1). The aim of the investigation was to chemically characterize aerial flowering parts of this plant: the dried methanol extract (TM), its alkaloid (AF) and non-alkaloid (NAF) fractions, and to assess their ability and the ability of detected flavonoids to inhibit enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), in vitro in the Ellman spectrophotometric test. Plant material was collected in eastern Serbia (Jelašnička Klisura gorge), dried, powdered and extracted with methanol, after dichloromethane pre-extraction. Using solvents of different polarities and pH value, AF (yield 14.35%) and NAF (yield 57.78%) were obtained from TM. By LC-MS analysis, flavonoids luteolin-8-C- glucosylpentoside (159.81 mg/g), genistin (22.38 mg/g), vitexin (6.32 mg/g), rutin (4.25 mg/g), and genistein (0.86 mg/g) were detected and quantified in TM, while luteolin-8-C- glucosylpentoside (212.70 mg/g), rutin (1.38 mg/g), and genistin (1.03 mg/g) in NAF. Using GC-FID-MS analysis, quinolizidine alkaloids sparteine, 17-oksosparteine, and lupanine were identified in AF. TM and NAF exhibited significant and comparable anti-cholinesterase activity (IC50AChE 0.54 and 0.72 mg/mL; IC50BuChE 0.45 and 0.68 mg/mL). All detected flavonoids evinced the ability to inhibit enzymes; the most active were genistein and vitexin (IC50AChE 54.75 and 88.35 μg/mL; IC 50BuChE 27.83 and 49.77 μg/mL). Considering the quinolizidine alkaloids toxicity and AF poor anti-cholinesterase activity, it might be concluded, regarding safety profile and medical potential, that the fraction of the herb methanol extract, flavonoid-rich and alkaloid-free (NAF), is good candidate for further research.Chamaecytisus heuffelii subsp. jankae (Velen.) Niketić (Fabaceae) je polužbun, koji raste na području istočnog dela Balkanskog poluostrva (Albanija, Bugarska, Srbija) (1). Cilj istraživanja bio je da se izvrši hemijska karakterizacija nadzemnih delova u cvetu ove biljke: suvog metanolnog ekstrakta (TM), njegove alkaloidne (AF) i frakcije bez alkaloida (NAF), i da se in vitro, u Ellmanov-om spektrofotomerijskom testu, ispita njihova sposobnost, kao i sposobnost detektovanih flavonoida da inhibiraju enzime acetilholinesterazu (AChE) i butirilholinesterazu (BChE). Biljni materijal je sakupljen u istočnoj Srbiji (Jelašnička klisura), osušen, samleven i ekstrahovan metanolom, nakon pre-ekstrakcije dihlormetanom. Primenom rastvarača različite polarnosti uz promenu pH vrednosti, iz TM su dobijene AF (prinos 14,35%) i NAF (prinos 57,78%). LC-MS analizom, u TM detektovani su i kvanitikovani flavonoidi luteolin-8-C-glukozilpentozid (159,81 mg/g), genistin (22,38 mg/g), viteksin (6,32 mg/g) i rutin (4,25 mg/g) i genistein (0,86 mg/g), a u NAF luteolin-8-C- glukozilpentozid (212,70 mg/g), rutin (1,38 mg/g) i genistin (1,03 mg/g). GC-FID-MS analizom, u AF identifikovani su hinolizidinski alkaloidi spartein, 17-oksospartein i lupanin. TM i NAF ispoljili su značajnu i uporedivu antiholinesteraznu aktivnost (IC50AChE 0,54 i 0,72 mg/mL; IC 50BChE 0,45 i 0,68 mg/mL). Svi detektovani flavonoidi su pokazali sposobnost da inhibiraju enzime; najaktivniji su bili genistein i viteksin (IC 50AChE 54,75 i 88,35 μg/mL; IC 50BuChE 27,83 i 49,77 μg/mL). Uzimajući u obzir toksič nost hinolizidinskih alkaloida i slabu antiholinesteraznu aktivnost AF, mož e se zaključ iti da je frakcija metanolnog ekstrakata herbe bogata flavonoidima i oslobođena alkaloida (NAF), u pogledu bezbednosnog profila i lekovitog potencijala dobar kandidat za dalja ispitivanja.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Preliminary analysis of specialised metabolites of Cytisus jankae flowering aerial part

Cytisus jankae Velen. (Fabaceae) is a dwarfish shrub (10–25 cm height) with 3-foliolate leaves and capitate inflorescence with tubular calyces. The whole plant is densely covered with silky hairs. Its range includes central and east part of the Bal- kan Peninsula and Romania. It inhabits dry rocky places on limestone or ultramafic soils. The objective of this study was to establish the phytochemical profile of flowering aerial parts of this plant. The plant material was collected in eastern Ser- bia (Jelašnička Klisura gorge). In the air-dried herb, by the direct gravimetric method, the total alkaloids content (0.32%) was determined, and using colorimetric assays the total poly- phenols (43.8%), tannins (1.13%), and the total flavonoids (1.31%) were quantified. Additionally, the powdered dried plant material was successively extracted with dichlorometh- ane (maceration) and methanol (bimaceration) at room tem- perature. After the solvents were evaporated under reduced pressure, dried extracts were subjected to the further analy- sis. The total alkaloids content in both the dichloromethane (7.01%) and methanol (3.47%) extracts, and the contents of total polyphenols (13.75%), tannins (2.45%) and flavonoids (4.65%) in the methanol extract, were determined by afore- mentioned tests. Applying the LC-MS method, in the metha- nol extract flavonoids quercetin 3-O-rutinoside, luteolin, api- genin and genistein were identified. GC-MS analysis resulted in the identification of quinolizidine alkaloids sparteine, 17-oxosparteine and lupanine (2-oxosparteine) in both the methanol and dichloromethane extracts, as well as of one tri- terpene, β-amyrin, in the dichloromethane extract. This is the first report on specialised metabolites from flowering aerial parts of C. jankae.7th Balkan Botanical Congress, 10-14th September 2018, Novi Sad, Serbi

Chemosystematic significance of triterpenes from dichloromethane extracts of 28 Hieracium L. species from the Balkan Peninsula

The genus Hieracium L. s. str. (Cichorieae, Asteraceae) is one of the most taxonomically complex genera of flowering plants. Chemosystematic significance of triterpenes especially for Asteraceae genera is scarcely assessed. The subject of this research are 28 Hieracium species from Balkan Peninsula (mostly collected at Mt Durmitor, Montenegro): 12 principal belonging to sections Pannosa, Naegeliana, Drepanoidea and Villosa, and 16 hybridogenous originated from the species from those four sections and Glauciformia, Hieracium, Prenanthoidea and/or Italica. Previously, in dried dichloromethane extracts of flowering aerial plant parts, α-amyrin and α-amyrin acetate, as well as β-amyrin, β-amyrin acetate and/or lupeol acetate were identified and quantified using GC-FID-MS. The aim was to evaluate chemosystematic significance of these five triterpenes using multivariate statistical methods (principal components analysis-PCA and non-metric multidimensional scaling-nMDS). Statistical analysis supported the current taxonomical classification of the investigated species, despite overlapping of certain groups. In PCA, all five triterpenes significantly contributed to the differences between the species. Moreover, lupeol acetate could be a significant chemosystematic marker for the most of principal species belonging to the section Pannosa, as well as for some hybridogenous species between H. gymnocephalum Gris. ex Pant. and the members from the other sections.14th Symposium on the Flora of Southeastern Serbia and Neighboring Regions, Kladovo, 26th to 29th June 202

Chemical composition and antioxidant potential of Teucrium scorodonia L. (Lamiaceae) from Serbia

The wood-sage (woodland germander) Teucrium scorodonia L. is widespread in European Atlantic coast and in the western part of Central Europe, with some isolated populations in Mediterranean. Recently discovered population on the territory of Serbia, in the valley of Štavica river (vicinity of Loznica), represents the most eastern enclave in the species range. Preliminary chemical analysis of aerial flowering parts and its dried ethanol and hydroethanol (70% v/v) extracts, and assessment of antioxidant potential of selected (hydroethanol) extract were performed. Using appropriate spectrophotometrical methods, total polyphenols in herb (3.48%, expressed as pyrogallol), ethanol and hydroethanol extracts (100.05 and 96.65 mg gallic acid equivalents - GAE/g), tannins in herb (0.96%, expressed as pyrogallol), ethanol and hydroethanol extracts (11.86 and 12.31 mg GAE/g), flavonoids in herb, ethanol and hydroethanol extracts (0.19%, 0.83% and 0.74%, expressed as hyperoside) were quantified. Also, in ethanol and hydroethanol extracts dihydroxycinnamic acid derivatives were determined (8.96% and 8.37%, expressed as acteoside). By LC-MS, in selected (hydroethanol) extract, four flavonoid and five phenylethanoid glycosides were detected; verbascoside (acteoside) was identified and quantified (4.42%) using external standard. This extract exhibited significant total antioxidant activity (1.37 mmol Fe2+/g) and moderate anti-DPPH potential (SC50=51.28 μg/mL).14th Symposium on the Flora of Southeastern Serbia and Neighboring Regions, Kladovo, 26th to 29th June 202

Investigation of triterpenes in the dichloromethane extracts of 28 Hieracium L. species (Asteraceae) from the Balkan peninsula

Rod Hieracium L. s. str. (Asteraceae) predstavlja jedan od najbrojnijih i najkompleksnijih rodova skrivenosemenica. Na Balkanskom poluostrvu, najveći broj vrsta ovog roda zabeležen je na Dinaridima, naročito na Durmitoru u Crnoj Gori. Cilj ovog istraživanja bio je da se utvrdi i međusobno uporedi profil triterpenskih jedinjenja u dihlormetanskim ekstraktima nadzemnih delova u cvetu 28 vrsta ovog roda poreklom iz Crne Gore i Srbije, i to: H. gymnocephalum Griseb. ex Pant., H. orieni A. Kern., H. blecicii Niketić, H. paratrichum Niketić, H. spirocaule Niketić, H. mokragorae (Nägeli & Peter) Nägeli & Peter, H. pannosum Boiss. s.l., H. plumulosum A. Kern., H. villosum Jacq., H. pilosum Froel., H. pseudoschenkii (Rohlena & Zahn) Niketić, H. naegelianum Pančić, H. anastrum (Degen & Zahn) Niketić, H. calophyllum R. Uechtr., H. scheppigianum Freyn, H. durmitoricum (Rohlena & Zahn) Niketić, H. guentheri‐beckii Zahn, H. mirificissimum Rohlena & Zahn, H. coloriscapum Rohlena & Zahn, H. pyricephalum Niketić, H. albopellitum (Zahn) Niketić, H. glabratum Willd., H. scorzonerifolium Vill. s.l., H. dentatum Hoppe s.l., H. valdepilosum Vill. s.l., H. neilreichii Beck, H. tommasinianum K. Malý i H. macrodontoides (Zahn) Zahn. U suvim dihlormetanskim ekstraktima nadzemnih delova biljaka u cvetu vršena je komparativna kvalitativna i kvantitativna GC‐FID‐MS analiza triterpena. Struktura triterpena utvrđena je poređenjem sa komercijalnim standardima, kao i na osnovu 1D i 2D NMR spektara odgovarajućih frakcija. U ispitivanim vrstama identifikovano je ukupno pet triterpena, i to dva derivata ursana (α‐amirin i njegov acetat), dva derivata oleana (β‐amirin i njegov acetat) i jedan derivat lupana (lupeol‐acetat). Dominantni triterpeni, u većini ispitivanih vrsta, bili su derivati ursana, a praćeni su jedinjenjima tipa oleana. U ovom radu je, za većinu (26) odabranih biljnih vrsta, ispitivanje triterpena sprovedeno po prvi put. Na osnovu dobijenih rezultata, značajno su upotpunjeni podaci o prisustvu i kompoziciji triterpenskih jedinjenja u vrstama roda HieraciumThe genus Hieracium L. s. str. (Asteraceae) represents one of the most numerous and most complex genera of flowering plants. In the Balkan Peninsula the largest number of species of this genus are recorded on Mt Durmitor in Montenegro. The objective of this study was to determine and compare triterpene profile of flowering aerial parts dichloromethane extracts of 28 Hieracium species from Montenegro and Serbia: H. gymnocephalum Griseb. ex Pant., H. orieni A. Kern., H. blecicii Niketić, H. paratrichum Niketić, H. spirocaule Niketić, H. mokragorae (Nägeli & Peter) Nägeli & Peter, H. pannosum Boiss. s.l., H. plumulosum A. Kern., H. villosum Jacq., H. pilosum Froel., H. pseudoschenkii (Rohlena & Zahn) Niketić, H. naegelianum Pančić, H. anastrum (Degen & Zahn) Niketić, H. calophyllum R. Uechtr., H. scheppigianum Freyn, H. durmitoricum (Rohlena & Zahn) Niketić, H. guentheri‐beckii Zahn, H. mirificissimum Rohlena & Zahn, H. coloriscapum Rohlena & Zahn, H. pyricephalum Niketić, H. albopellitum (Zahn) Niketić, H. glabratum Willd., H. scorzonerifolium Vill. s.l., H. dentatum Hoppe s.l., H. valdepilosum Vill. s.l., H. neilreichii Beck, H. tommasinianum K. Malý and H. macrodontoides (Zahn) Zahn. In dried dichloromethane extracts of flowering aerial plants parts comparative qualitative and quantitative GC‐FID‐MS analysis of triterpenes was performed. The structures of triterpenes were elucidated comparing to commercial standards, and on the basis of 1D and 2D NMR spectra of corresponding fractions. In investigated species, overall five triterpenes, including two of ursane (α‐amyrin and its acetate), two of oleane (β‐amyrin and its acetate) and one of lupane (lupeol acetate) type, were identified. Dominant triterpenes, for the majority of species, were of ursane type, followed by representatives of oleane type. In this paper, for the majority (26) of selected plant species, triterpenes were tested for the first time. Based on obtained results, the data on the presence and composition of triterpen compounds in Hieracium species have been significantly supplemented.VII Kongres farmaceuta Srbije sa međunarodnim učešćem: Zajedno stvaramo budućnost farmacije, Beograd, Srbija, 10-14. oktobar 2018.Saopštenje sa skupa nacionalnog značaja štampano u izvod

From Aloe vera Leaf Waste to the Extracts with Biological Potential: Optimization of the Extractions, Physicochemical Characterization, and Biological Activities

In the study, the optimization of the extraction from Aloe vera leaf waste was performed via varying solid-to-solvent ratio, solvent type, extraction time, and technique (maceration, heat-, ultrasound-, and microwave-assisted extractions—HAE, UAE, and MAE, respectively). The optimal extraction conditions for achieving the highest polyphenol content are a 1:30 ratio, 70% ethanol, and 30 min of HAE. Total flavonoid and protein contents were significantly higher in the extract from MAE, while total condensed tannin content was the highest in HAE. LC-MS analysis quantified 13 anthraquinone and chromone compounds. The variations in the FT-IR spectra of the extracts obtained by different extraction procedures are minor. The influence of extraction conditions on the antioxidant ability of the extracts depended on applied antioxidant assays. The extracts possessed medium inhibition properties against Staphylococcus aureus and weak inhibitory activity against Enterococcus feacalis. The extracts had stimulative effect on HaCaT cell viability. Regarding the extraction yield, there was a significant difference between the used extraction techniques (MAE > HAE > maceration and UAE). The presented study is an initial step in the production of polyphenol-rich extracts from A. vera leaf waste aimed to be used for the potential preparation of pharmaceutical and cosmetic formulations for the skin

Antiradical activity and phenolic constituents of Stachys recta L. subsp. recta herb methanol extract

The highest richness of taxa within genus Stachys was recorded in the wider area of the Mediterranean, where the group S. recta stands out in terms of taxonomic complexity. The center of diversity of this group is on the Balkan Peninsula. Its representatives are still insufficiently defined in taxonomic and horological terms, and their traditional approach includes about fifteen taxa, mostly at the infraspecific level. Despite some radical approaches that were presented at the end of the last century [1], the traditional taxonomic treatment is still prevailed in the relevant global lists. In this work antiradical potential and phenolic constituents of S. recta L. subsp. recta were investigated. Plant material, collected in Central Serbia (vicinity of Prokuplje), was air-dried, powdered, and then extracted by bimaceration with chloroform and methanol. Obtained dry methanol extract exhibited strong, concentration-dependant anti-DPPH activity (SC50=8.23 μg/mL), with total phenolics content at 0.27 mg GAE/mg of dry extract. In TLC-DPPH test observed yellow zones indicated presence of few compounds which strongly neutralized purple DPPH radical. LC-MS analysis revealed presence of 24 phenolic compounds, belonging to three classes of constituents: phenolic acids, phenylethanol glycosides and flavonoids. Chlorogenic acid (2.63%) was the only phenolic acid, while acteoside was the most dominant phenylethanoid (24.03%). Flavonoid fraction was composed almost entirely of 8-hydroxyflavones, derivatives of hypolaetine and isoscutellareine (53.47%). The most prominent flavonoids in investigated extract were 7-O-[6'''-acetylallosyl(1®2)]-glucopyranosides of 4'-O-methylhypolaetine and hypolaetine (15.92 and 13.01%, respectively).5th International Conference on Natural Products Utilization: from Plants to Pharmacy Shelf - ICNPU-2023, Sts. Constantine & Helena, Bulgaria, 30th May to 2nd June 202

Antiradikalska aktivnost i fenolni sastojci metanolnog ekstrakta herbe Stachys milanii

Stachys milanii Petrović (Lamiaceae) is an endemic of central and eastern part of Balkan Peninsula, growing mainly in river valleys on semi-saline soils, often along the edge of cereal fields and abandoned arable lands. It is a critically endangered and strictly protected species in Serbia, with just one proven habitat (1). So far it was investigated on herb essential oil composition and antimicrobial activity, and on fatty acid composition of seeds (2). Aim of this work was to investigate antiradical potential and phenolic constituents of dry methanol herb extract. Aerial flowering parts (herb) of S. milanii were collected in North Macedonia (Ovče Pole). Powdered, dried plant material was extracted by bimaceration with chloroform, and then with methanol. Obtained dry methanol extract was used for further analysis. Using appropriate spectrophotometric procedure strong, concentration-dependant anti-DPPH activity of extract was observed (SC50=34.15 μg/mL). Also spectrophotometrically, using Folin-Ciocalteu reagent, significant total phenolics content (0,47 mg gallic acid/mg dry extract) was determined. In TLC-DPPH test observed yellow zones indicated presence of few compounds which strongly neutralized purple DPPH radical. LC-MS analysis revealed presence of one phenolic acid, four phenylethanol glycosides, eight flavonoid glycosides derivatives of 8-hydroxyflavones, and one apigenin glycoside. Using commercial standards in extract were identified and quantified: chlorogenic acid (1.63%) and acteoside (2.65%), as the main phenylethanoid. In 8-hydroxyflavones fraction six hypolaetine and two isoscutellarein derivatives were present; using compounds previously isolated from S. alpina subsp. dinarica Murb. herb, 4'-O-methylhypolaetine-7-O-[6'''-acetylallosyl(1®2)]-glucopyranoside and isoscutellarein-7-O-[6'''-acetylallosyl(1®2)]-glucopyranoside were identified, representing the most prominent flavonoids in investigated extract, respectively. References 1. Niketić M, Diklić N. Stachys milanii Petrović. In: Stevanović V. (Ed.) Red Book of Flora of Serbia 1 - Extinct and Critically Endangered Taxa. Belgrade: Ministry for the Protection of Natural Resources and the Environment, Faculty of Biology, University of Belgrade, Institution for Protection of Nature of the Republic of Serbia. 1999; pp. 211-3, 466-7. 2. Stojanovic G, Ligon AP, Smelcerovic A, Lazarevic J, Spiteller M, Palic R. Fatty acids of Stachys milanii seeds. Chem Nat Comp 2007;43(4):380-3. Acknowledgements This research was funded by the Ministry of Education, Science and Technological Development, Republic of Serbia through Grant Agreement with University of Belgrade-Faculty of Pharmacy No: 451-03-68/2022-14/200161.Stachys milanii Petrović (Lamiaceae) je endemit centralnog i istočnog dela Balkanskog poluostrva koji raste pretežno u dolinama reka na poluslanim zemljištima, često i po obodu žitnih polja i po napuštenim oranicama. U Republici Srbiji je kritično ugrožena i strogo zaštićena vrsta, sa samo jednim zabeleženim staništem (1). Biljka je do sada ispitivana u pogledu sastava i antimikrobne aktivnosti etarskog ulja herbe i kompozije masnih kiselina semena (2). Cilj ovog rada bio je da se ispitaju antiradikalski potencijal i fenolni sastojci suvog metanolnog ekstrakta herbe. Nadzemni delovi S. milanii u cvetu prikupljeni su u Severnoj Makedoniji (Ovče Pole). Sprašen, osušen biljni materijal je ekstrahovan posupkom bimaceracije najpre hloroformom, a zatim metanolom. Dobijeni suvi metanolni ekstrakt korišćen je za dalju analizu. Odgovarajućim spektrofotometrijskim postupkom utvrđena je snažna, koncentraciono-zavisna anti-DPPH aktivnost ekstrakta (SC50=34,15 μg/mL). Spektrofotometrijski, korišćenjem Folin-Ciocalteu reagensa, utvrđen je i značajan sadržaj ukupnih polifenola (0,47 mg galne kiseline/mg suvog ekstrakta). U TLC-DPPH testu uočene žute zone ukazale su na prisustvo većeg broja jedinjenja koja snažno neutrališu ljubičasti DPPH-radikal. LC-MS analizom u ekstraktu su detektovani jedna fenolkarboksilna kiselina, četiri feniletanska heterozida, osam flavonoidnih heterozida derivata 8-hidroksiflavona i jedan heterozid apigenina. Korišćenjem komercijalnih standarda identifikovni su i kvantifikovani hlorogenska kiselina (1,63%) i akteozid (2,65%), kao glavni feniletanski heterozid. Frakciju derivata 8-hidroksiflavona činilo je šest heterozida hipolaetina i dva heterozida izoskutelareina; korišćenjem jedinjenja prethodno izolovanih iz herbe S. alpina subsp. dinarica Murb., identifikovani su 4'-O-metilhipolaetin-7-O-[6'''-acetilalozil(12)]- glukopiranozid i izoskutelarein 7-O-[6'''-acetilalozil(12)]-glukopiranozid, koji su ujedno predstavljali i najzastupljnije flavonoide u ekstraktu, redom.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra