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With regard to world’s increasing demand for biologically active compounds, a novel source of xanthones and secoiridoid glycosides has been studied . Zeltnera beyrichii (Torr. & A. Gray) Mans., an insufficiently acknowledged North American medicinal plant species, may be considered a pharmacological substitute for commercial C. erythraea Rafn, since it accumulates in aerial parts nearly the same amount of secoiridoid glycosides: swertiamarin, gentiopicrin, and sweroside (13.76, 7.56, and 0.17 mmol per 100 g dry weight, respectively) in plants grown under greenhouse condition, and a considerable amount of xanthones: decussatin and eustomin. Additionally, Z. beyrichii produced as much biomass during cultivation in RITA ® temporary immersion bioreactors as greenhouse-grown plants, in a third of the time. Plants grown in bioreactors contained moderate levels of total phenolics and total flavonoids, and possessed modest antioxidant activity and antimicrobial potential against eight bacterial and eight fungal species. Therefore, this species may be highly recommended for cultivation either in natural environment, or in bioreactors under in vitro conditions, for producing compounds of interest of modern pharmacology and food industry.

We studied the allelopathic potential of Nepeta rtanjensis Diklic and Milojevic essential oil and pure alpha- and beta-pinene on seed germination of 4-crops (Lepidium sativum L., Lactuca sativa L. cv. 'May Queen', Lotus corniculatus L. cv. Bokor and Brassica napus L. cv. napus) and 3-weed species (Stellaria media (L.) Vill., Rumex crispus L. and Arabidopsis thaliana (L.) Heynh. ecotype Columbia). Nepetalactones are dominant compounds in essential oil of N. rtanjensis (88%), while alpha- and beta-pinene were present in low concentrations (3% and 0.4%, respectively). We found the species-specific and dose-dependent effects of essential oils and pure compounds on seed germination of test species. Among tested crops, L. sativa proved to be the most sensitive, while B. napus was most tolerant to both essential oil and pure monoterpenoids. S. media weed was the most susceptible, while R. crispus was most tolerant. Due to its high allelopathic potential, of nepetalactones, N. rtanjensis essential oil could be developed as promising natural bioherbicide to control weeds.

Introduction - Nepeta species contain a variety of secondary metabolites, including iridoid monoterpenes - nepetalactones and phenolic acids - that are considered the main bioactive constituents. This work represents the first attempt to comparatively explore variations in these two major groups of secondary metabolites within the genus. Objective - To develop an efficient analytical methodology for simultaneous analysis of nepetalactones and phenolic acids in methanol extracts of selected Nepeta species, and to evaluate its potential application in chemotaxonomic studies. Material and methods - A UHPLC combined with linear-trap quadrupole (LTQ) orbitrap MS method was used to characterise chemical diversity and complexity of phenolics among 12 selected Nepeta species. A targeted metabolomic approach using UHPLC coupled to a diode array detector (DAD) and combined with (+/-) heated electrospray ionisation (HESI) MS/MS was developed and validated for quantitative analysis of six hydroxycinnamic acid derivatives and four nepetalactones. Results - Phenolic profiling provided a valuable database of bioactive compounds in the plant group studied, including phenolic acids (hydroxybenzoic and hydroxycinnamic acids) and flavonoids (flavones, flavonols and flavanones). Principal component analysis and cluster analysis suggested the applicability of 10 targeted compounds as chemomarkers for chemotaxonomic studies. Pearson's correlation analysis revealed significant positive correlations between metabolites involved in different biosynthetic pathways (phenylpropanoid or monoterpenoid). Conclusion - The described targeted metabolomic approach proved to be highly beneficial in designing a phytochemical overview of the genus Nepeta, and might have applications in further clarification of phylogenetic relations. Furthermore, it has the potential to be implemented in a routine quality control of plant material and herbal preparations. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3420]

Nepeta rtanjensis Diklić i Milojević, N. sibirica L. i N. nervosa Royle ex Bentham (fam. Lamiaceae) su endemične vrste roda Nepeta, koje su nedovoljno fitohemijski okarakterisane i slabo ispitane u pogledu biološke aktivnosti. Radi se o vrstama koje se odlikuju različitim kvalitativnim i kvantitativnim sadržajem nepetalaktona, zbog čega predstavljaju idealne objekte u istraživanjima biološke aktivnosti različitih izomera ovog monoterpenskog jedinjenja. S obzirom da se radi o retkim vrstama, a u slučaju N. rtanjensis i o krajnje ugroženoj vrsti, biljni materijal za potrebe eksperimenata je dobijen mikropropagacijom. Istraživanje je obuhvatilo fitohemijske analize sekundarnih metabolita tri vrste roda Nepeta gajenih in vitro, kao i analizu njihovih bioaktivnih svojstava kao što su antimikrobno, antioksidativno i alelopatsko dejstvo. Fitohemijska karakterizacija tri vrste roda Nepeta gajenih in vitro, omogućila je identifikaciju i kvantifikaciju glavnih grupa sekundarnih metabolita (terpenoida i fenolnih jedinjenja) koji se u ovim uslovima produkuju. Metode koje su korišćene u fitohemijskim analizama su: GC-MS, GC-FID, PTR-MS, Headspace GC-MS, HPLCUV, HPLC-MS, UHPLC/DAD/±HESI-MS/MS. Dominantno isparljivo jedinjenje iz grupe terpenoida je monoterpenski lakton - nepetalakton, pri čemu je kod vrste N. rtanjensis većinski prisutan tran,cis- izomer a kod N. sibirica cis,trans- izomer nepetalaktona. Kod vrste N. nervosa nepetalakton je detektovan samo u tragovima. Kod ispitivanih vrsta, ruzmarinska kiselina se javlja kao dominantno fenolno jedinjenje, dok su ostale fenolne kiseline (hlorogena, neohlorogena i kafeinska kiselina) prisutne u znatno nižoj koncentraciji. Metanolni ekstrakti N. rtanjensis, N. sibirica i naročito N. nervosa, pokazuju značajnu antioksidativnu aktivnost u ABTS i DPPH testovima, kao i visoki kapacitet za redukciju gvožđa u FRAP testu. Značajna antioksidativna aktivnost se pripisuje fenolnim jedinjenjima, na prvom mestu ruzmarinskoj kiselini. Nepetalakton ne utiče značajno na antioksidativni potencijal metanolnih ekstrakata, ali postoji mogućnost antagonističkog delovanja između fenolnih kiselina i nepetalaktona. Metanolni ekstrakti tri vrste roda Nepeta pokazuju značajnu antimikrobnu aktivnost protiv osam vrsta bakterija i osam vrsta gljiva, a aktivnost potiče od monoterpenskih laktona i fenolnih jedinjenja. Najveću antimikrobnu aktivnost pokazuje metanolni ekstrakt N. rtanjensis, sledi N. sibirica, i na kraju N. nervosa. Stereohemija nepetalaktona bitno određuje njegovu antimirobnu aktivnost, pri čemu je trans,cisizomer aktivniji od cis,trans-izomera. Potvrđeno je da vrste N. rtanjensis i N. sibirica poseduju jedinjenja koja mogu uticati na rastenje i razviće drugih biljnih vrsta u njihovom neposrednom okruženju, i na taj način aktivno učestvovati u alelopatskim interakcijama. Alelopatsko dejstvo N. rtanjensis i N. sibirica potiče od dominantnog bioaktivnog jedinjenja ovih vrstanepetalaktona. Stereohemija nepetalaktona značajno određuje njegov alelopatski potencijal, pri čemu tran,cis- izomer pokazuje veću aktivnost od cis,trans- izomera. Alelopatsko dejstvo nepetalaktona se ogleda kroz usporenu dinamiku klijanja semena test vrste Lepidium sativum L., ali i kroz efekat na biohemijske procese koji su posledica poremećenog antioksidativnog sistema biljaka. Alelopatsko dejstvo nepetalaktona, a naročito njegovog trans,cis- izomera, dovodi do promena u antioksidativnom sistemu klijanaca kresa narušavanjem normalnih modela ekspresije i aktivnosti antioksidativnih enzima (POD, CAT, SOD, PPO). Primećena je inhibicija aktivnosti, kao i izmenjen profil izoformi POD, CAT, Fe-SOD i CuZn-SOD kod klijanaca kresa. Trans,cisnepetalakton dovodi do povećanog sadržaja ukupnih fenola u prvim fazama rastenja i razvića klijanaca L. sativum, što je praćeno ranijom indukcijom ekspresije i povećanom aktivnošću PPO. Nepetalakton tokom alelopatskih interakcija indukuje određene modifikacije metabolizma sinapata kod klijanaca kresa, koje se generalno mogu opisati usporenim prevođenjem sinapoil- holina do sinapoil- glukoze, i potom do sinapoilmalata. Alelopatska svojstva N. rtanjensis i N. sibirica ukazuju na mogućnost uspešne primene ovih vrsta i njihovih bioaktivnih komponenti u poljoprivrednoj praksi, kao bioherbicida, a u cilju suzbijanja i kontrole korovskih vrsta. U ovom radu potvrđeno je alelopatsko dejstvo N. rtanjensis i N. sibirica u slučaju poljoprivrednih kultura kao što su L. sativum, Lactuca sativa L. sorta „Majska kraljica“, Lotus corniculatus L. sorta „Bokor“, Brassica napus L., i korovskih vrsta poput Stellaria media (L.) Vill., Rumex crispus L., i Arabidopsis thaliana (L.) Heynh. ekotip Kolumbija. Nepeta rtanjnsis Diklić and Milojević, N. sibirica L. and N. nervosa Royle ex Bentham (fam. Lamiaceae) are endemic species of genus Nepeta that are insufficiently phytochemically characterized and poorly tested in a sense of their biological activity. These three species are characterized by different qualitative and quantitative nepetalactone content, what makes them ideal objects in investigating the biological activity of different stereoisomers of this compound. Considering that these species are rare, and extremely endangered in case of N. rtanjensis, plant material for the experiments was obtained by micropropagation. The research included phytochemical characterization of in vitro grown three Nepeta species , as well as analysis of their bioactivities such as antimicrobial, antioxidative activity and allelopathic potential. Phytochemical characterization of three Nepeta species grown in vitro, involving GC-MS, GC-FID, PTR-MS, Headspace GC-MS, HPLC-UV, HPLC-MS, and UHPLC/DAD/±HESI-MS/MS analyses, enabled identification and quantification of the main secondary metabolites (terpenes and phenolics). The dominant volatile compound from terpenes group is monoterpene lacton - nepetalactone, whereby its trans,cisisomer is mostly present in N. rtanjensis and its cis,trans- isomer in N. sibirica. In N. nervosa, nepetalactone has been detected only in traces. In all investigated species, rosmarinic acid is a dominant phenolic compound, while the other phenolic acids (chlorogenic, neochlorogenic and caffeic acids) are present in significantly lower concentrations. The results of ABTS and DPPH assays showed that methanol extracts of N. rtanjensis, N. sibirica and especially N. nervosa, possess considerable antioxidant activities, and the FRAP assay revealed high ferric reducing capacity for all the samples tested.. Significant antioxidant activity is attributed to phenolic acids, in the first place to rosmarinic acid. Nepetalactone doesn’t contribute notably to the antioxidant potential of the methanol extracts, but there is a possibility of antagonistic action between phenolic acids and nepetalactone. The methanol extracts of three Nepeta species show significant antimicrobial activity against eight bacterial and eight fungal species, and the activity originates from monoterpenic lactones and phenolic acids. The highest antimicrobial activity was recorded for N. rtanjensis methanol extract, while extracts of N. sibirica and N. nervosa were less efficient. Nepetalactone stereochemistry substantially determines its antimicrobial activity, whereby the trans,cis- isomer is more active than the cis,trans- one. It has been confirmed that N. rtanjensis and N. sibirica possess compounds that can influence growth and development of other plant species in their proximate surrounding, thus actively participating in allelopathic interactions. Allelopathic effect of N. rtanjensis and N. sibirica originates from the dominant bioactive compound of these species - nepetalactone. Stereochemistry of nepetalactone considerably determines its allelopathic potential, with trans,cis- isomer being more active than the cis,transnepetalactone. Allelopathic effect of nepetalactone is reflected through slugged dynamic of seed germination in the test species Lepidium sativum L., but also through its effect on biochemical processes that are the consequence of plant’s disturbed antioxidative system. Allelopathic effect of nepetalactone, especially of its trans,cis- isomer, leads to the changes in antioxidative system of cress seedlings by violating normal models of antioxidative enzymes expression and activity (POD, CAT, SOD, PPO). In cress seedlings, the inhibition of activities and changed profiles of POD, CAT, Fe-SOD and CuZn-SOD isoforms were observed. Trans,cis- nepetalactone action leads to the increased content of total phenols in the first phases of growth and development of L. sativum seedlings, which is followed by earlier induction of expression and increased PPO activity. During allelopathic interactions, nepetalactone induces certain modifications of sinapate metabolism in cress seedlings, which in general can be described by decreased conversion of sinapoylholin to sinapoylglucose, and further to sinapoylmalate. Allelopathic features of N. rtanjensis and N. sibirica indicate the possibility of successful application of these species and their bioactive components in agricultural practice, as bioherbicides, toward weed control. In this thesis the allelopathic effect of N. rtanjensis and N. sibirica was confirmed in case of crops L. sativum, Lactuca sativa L. cv. „Majska kraljica“, Lotus corniculatus L. cv. „Bokor“, and Brassica napus L., and weeds Stellaria media (L.) Vill., Rumex crispus L., and Arabidopsis thaliana (L.) Heynh. ecotype Colombia.