Chemical and pharmacological characterisation of the essential oils of the plant species Seseli gracile Waldst. and Kit. and Seseli pallasii Besser (Apiaceae)

У оквиру ове докторске дисертације извршена је хемијска и фармаколошка карактеризација етарских уља изолованих из две врсте рода Seseli: Seseli pallasii и Seseli gracile. У испитивању су коришћени биљни материјали S. pallasii (корен, ризом, херба и плод) и S. gracile (корен, херба, цваст и плод) прикупљени током шестогодишњег периода са природног станишта као и гајени (само S. gracile). Етарска уља су изолована дестилацијом воденом паром, након чега су хемијски анализирана методом гасне хроматографије уз масени (GC-MS) и пламено-јонизујући детектор (GC-FID). Резултати хемијске анализе су показали да у узорцима надземних органа обе испитиване врсте доминирају монотрепенска једињења, са најзаступљенијим компонентама α-пиненом (S. pallasii) и терпиноленом (S. gracile). У подземним органима обе врсте доминирају не-терпенска једињења: n-нонан и n-ундекан (S. pallasii), односно фалкаринол (S. gracile). Хемијске варијације у саставу етарских уља изолованих из различитих биљних делова су анализиране мултиваријантним статистичким методама (PCA и HCA). У оквиру фармаколошке карактеризације спроведена су in vitro испитивања антирадикалске (DPPH метода), антимикробне (микродилуциона метода), спазмолитичке (метода на изолованом илеуму пацова), вазорелаксантне (метода на изолованој мезентеричној артерији пацова), и ACE инхибиторне активности (колориметријска метода). Испитивању in vitro ACE инхибиторне активности, претходило је in silico испитивање, где је разматран и потенцијални утицај појединачних компоненти на инхибицију ACE. Тестирана етарска уља су показала добар спазмолитички и вазорелаксантни (S. pallasii) умерен антимикробни (S. pallasii) и антирадикалски ( S. gracile), а слаб ACE инхибиторни потенцијал.Chemical and pharmacological characterisation of essential oils isolated from two Seseli species: Seseli gracile and Seseli pallasii were explored in this doctoral dissertation. Samples of S. pallasii (root, rhizome, herb and fruit) and S. gracile (root, herb, inflorescence and fruit) collected from a natural habitat over a six-year period and cultivated plants (just for S. gracile) were used in this research. Essential oils were isolated from the plant material by hydro-distillation method, and were thereafter chemically analysed by gas chromatography method with mass (GC-MS) and flame-ionizing detector (GC-FID). The results of the chemical analysis suggest that essential oils isolated from aerial parts of both investigated species are dominated by monoterpenes with α-pinene (S. pallasii) and terpinolene (S. gracile) as the most abundant compounds. In essential oils from underground plant organs, non-terpenic compounds are predominantly present in both species: n-nonane and n-undecane (S. pallasii) and falcarinol (S. gracile). Chemical composition variations of essential oils isolated from different plant parts were statistically analyzed using multivariate statistical methods (PCA and HCA). Within the pharmacological characterization, in vitro studies of antiradical (DPPH test), antimicrobial (microdilution test), spasmolytic (isolated rat ileum method), vasorelaxant (isolated mesenteric artery method) and ACE inhibitor (colorimetric test) activities were conducted. In vitro test of ACE inhibitor activity was preceded by in silico test, where the individual compounds potential for ACE inhibition was analyzed. Tested essential oils exhibited good spasmolytic and vasorelaxant activity (S. pallasii), moderate antimicrobial (S. pallasii) and anti-DPPH (S. gracile) activity and low ACE inhibition potential

Антидијабетски потенцијал једноставних карбамата: компаративна експериментална и рачунарска студија

With the increasing global burden of diabetes mellitus type 2, the search for the new drugs, with better pharmacological profile is continued. As a part of this surge, the synthesis, pharmacological in vitro and computational evaluation of five, simple carbamate derivatives, against carbohydrate digestive enzyme α-glucosidase, is disclosed herein. Results of the experimental and computational assessment indicated that examined carbamates deterred the activity of α-glucosidase with acceptable IC50 values ranging from 65.34 to 79.89 μM compared to a standard drug acarbose (109.71 μM). Similarly, the studied compounds displayed in silico binding affinity for α-glucosidase enzyme with significant binding energies. Preliminary toxicity profiles of studied carbamates against three cancerous cell lines indicated their poor activity, suggesting that significant structural modifications have to be made to improve their anticancer efficiency. Results of the present study indicate that the examined carbamates were able to virtually or experimentally interact with an important target of diabetes mellitus type 2. Additionally, a new pharmacophore model is proposed featuring hydrogen bond donating carbamate –NH group, hydrogen bond accepting carbamate –OCH3 group and hydrophobic stabilization of aromatic moieties.Са порастом појаве дијабетеса типа 2 у свету, јавља се потрага за новим лековима са што ефикаснијим фармаколошким профилом. Као део овог истраживања, приказана је синтеза, фармаколошко in vitro и рачунарско испитивање пет карбамата једноставне структуре, као инхибитора – глукозидазе, ензима који учествује у дигестивном разлагању шећера. Резултати експерименталног испитивања показали су да испитивани карбамати инхибирају активност – глукозидазе са задовољавајућим IC50 вредностима у опсегу од 65,34 до 79,89 μM, а у поређењу са стандардним леком, акарбозом (109,71 μM). Такође, in silico методом добијене су значајне енергије везивања за активно место – глукозидазе. У прелиминарном испитивању цитотксичности према три типа канцерозних ћелија, карбамати су показали лошу активност, сугеришући да су потребне значајне структурне промене за побољшање њиховог антиканцерозног дејства. Уопштено говорећи, резултати ове студије показали су да су испитивани карбамати успешно виртуелно и експериментално интераговали са важном метом код дијабетеса типа 2. Такође је предложен и нови фармакофорни модел за -глукозидазу, који укључује карбаматну –NH групу као донора водоничне везе, затим карбаматну –OCH3 групу као акцептора водоничне везе, а такође и стабилизујуће хидрофобне интеракције ароматичних прстенова

Determination of arsenic content in tea samples available on Republic of Srpska market by atomic absorption spectrophotometry

Introduction. Arsenic exists in various forms in nature and living organisms.Toxic elements, including arsenic, which are present in some plants,can severely damage haemopoietic, immune, nervous and reproductivesystems. For this reason, a content of heavy metals is one of the criteria forthe assessment of the safe use of plant material in the production of traditionalmedicines and herbal infusions. This instigates the need for constantand organized safety control of plants that are used as raw materialsin pharmaceutical industry.The aim of this study is to determine the arsenic content in selected teaswhich are available on the market of the Republic of Srpska.Methods. The 10 g samples of 13 herbal and 3 fruit teas were mineralizedby dry ashing and arsenic contents were determined by the atomicabsorption spectrophotometer Agilent Technologies Series 200 with anair-acetylene burner and D2 background correction.Results. Mean arsenic concentrations in the herbal tea samples rangedfrom 0.009 to 0.145 mg/kg. The lowest arsenic concentration in a singlesample of 0.007 mg/kg was found in Chamomile tea and Uva ursi collectedas a wild plant at elevation above 1200 m. The highest arsenic concentrationwas found in the sample of Sambucus nigra tea (0.145 mg/kg). Infruit teas, the arsenic concentration ranged from 0.014 mg/kg (Cranberry)to 0.027 mg/kg (Fruit mix).Conclusion. Arsenic content in all analyzed tea samples is below the valuestipulated by the national legislation

Vasorelaxant activity of terpinolene

Terpeni predstavljaju veoma važnu grupu hemijskih jedinjenja kako zbog industrijske upotrebe, tako i zbog svojih značajnih bioloških efekata koji se mogu iskoristiti u medicini. Jedna su od najbrojnijih grupa sekundarnih metabolita biljaka, a naziv ove klase jedinjenja potiče od terpentina (terpentinskog ulja), tečnog proizvoda destilacije oleorezina bora. Terpinolen (p‐menta‐2,4(8)‐dien) je bezbojna ili bledo žuto obojena tečnost aromatičnog mirisa. Ovaj monociklični, monoterpenski alken je široko zastupljen u biljnim tkivima različitih četinara, paškanata, konoplje, čajevca, listu kurkume i peršuna, a čest je sastojak etarskog ulja Citrus, Mentha, Juniperus i Myristica vrsta. Jedna je od glavnih komponenata etarskog ulja nadzemnih delova endemične vrste Seseli gracile Waldst. & Kit. (6,1‐57,5 %). Terpinolen poseduje potvrđenu antiradikalsku, antihiperalgezijsku i antiedematoznu aktivnost. S obzirom da su neka strukturno slična monoterpenska jedinjenja pokazala vazorelaksantnu aktivnost, cilj ovog istraživanja je da se istraži vazorelaksantni potencijal terpinolena. U ovom istraživanju ispitivan je efekat standarda terpinolena (43905 Sigma‐ Aldrich) na izolovanoj renalnoj arteriji pacova Wistar soja. Korišćeni su prstenasti segmenti renalne arterije dužine 3‐5 mm sa očuvanim endotelom. Arterijski segmenti su postavljani u vodeno kupatilo u Krebs‐Ringerov rastvor, na 37C i aerisani kontinuiranim dovođenjem smješe 95 % O2 i 5 % CO2. Kontrakcija krvnih sudova je izazvana primjenom fenilefrina (10‐6 M). Integritet endotela je potvrđen farmakološki, primjenom acetilholina (10‐6 M). Na stabilan tonus krvnog suda izazvan fenilefrinom su dodavane rastuće koncentracije terpinolena, pripremljenog od standarda razblaživanjem u 5 % karboksi‐metil celuloze (0,2 % ‐ 33,3 %, kumulativno). Svaka sledeća koncentracija je dodavana tek nakon što se završi efekat prethodno primjenjene koncentracije, tj. otprilike nakon 15 minuta od prethodne. U kontrolnoj seriji eksperimenata je na isti način dodavan rastvarač (6 dodavanja kumulativno, sa vremenskim razmakom od 15 minuta). Utvrđeno je da terpinolen u kocentraciji od 11,1% dovodi do statistički značajne vazorelaksacije u odnosu na rastvarač kao kontrolu (F=2,584, p<0,05; Two‐way ANOVA). Naši rezultati opravdavaju nastavak ispitivanja potencijalne primene terpinolena u terapiji kardiovaskularnih oboljenja.Terpenes represent a very important group of chemical compounds because of their industrial use, as well as medical use which is possible due to their significant biological effects. They are one of the largest groups of secondary plant metabolites. The name of this class of compounds comes from turpentine (oil of turpentine), a liquid product of pine oleoresin distillation process. Terpinolene (p‐menth‐2,4 (8)‐diene) is a colorless or pale yellow colored liquid with aromatic odor. This monocyclic, monoterpenic alkene is widely represented in plant tissues of various pines, pashkans, cannabis, tea tree, turmeric and parsley leaves, and it is often a component of essential oils obtained from Citrus, Mentha, Juniperus and Myristica species. It is one of the main components of the essential oil of aerial parts of the endemic species Seseli gracile Waldst. & Kit. (6.1‐57.5%). Terpinolen possesses confirmed antiradical, antihyperalgesic and antiedematous activity. Since some structurally similar monoterpenic compounds have previously shown vasorelaxant activity, the aim of this study was to investigate the vasorelaxant potential of terpinolene. In the present study, the effects of standard terpinolene (43905 Sigma‐Aldrich) on the isolated renal artery of Wistar rats were investigated. Three to five mm long arterial ring tissue segments with preserved endothelium were used. The arterial segments were placed in water bath in the Krebs‐Ringer solution, at 37° C and continuously aerated by 95% O2 and 5% CO2. Contractions of the blood vessels were induced with 10‐6 M of phenylephrine. The endothelium integrity was confirmed pharmacologically, by using the acetylcholine (10‐6M). After accomplishing tonic phase of the contraction, increasing concentration of terpinolene (0.2 % ‐ 33.3 %, cumulatively) in 5 % carboxymethyl cellulose (CMC) solution were added to the organ bath. Each subsequent concentration was added only after the end of effect of the previously applied concentration, i.e. approximately 15 minutes from the previous one. In the control series of experiments, a solvent was added in the same manner. It was found that terpinolene at the concentration of 11.1% exibits statistically significant vasorelaxation in comparison to the solvent control (F = 2.584, p <0.05; Two‐way ANOVA). Our results justify a continuation for the further studies of terpinolene’s potential in the treatment of cardiovascular diseases.VII Kongres farmaceuta Srbije sa međunarodnim učešćem: Zajedno stvaramo budućnost farmacije, Beograd, Srbija, 10-14. oktobar 2018

In Silico and In Vitro Studies of Alchemilla viridiflora Rothm-Polyphenols' Potential for Inhibition of SARS-CoV-2 Internalization

Since the outbreak of the COVID-19 pandemic, it has been obvious that virus infection poses a serious threat to human health on a global scale. Certain plants, particularly those rich in polyphe- nols, have been found to be effective antiviral agents. The effectiveness of Alchemilla viridiflora Rothm. (Rosaceae) methanol extract to prevent contact between virus spike (S)-glycoprotein and angiotensin- converting enzyme 2 (ACE2) and neuropilin-1 (NRP1) receptors was investigated. In vitro results revealed that the tested samples inhibited 50% of virus-receptor binding interactions in doses of 0.18 and 0.22 mg/mL for NRP1 and ACE2, respectively. Molecular docking studies revealed that the compounds from A. viridiflora ellagitannins class had a higher affinity for binding with S-glycoprotein whilst flavonoid compounds more significantly interacted with the NRP1 receptor. Quercetin 3-(6”-ferulylglucoside) and pentagalloylglucose were two compounds with the highest exhibited interfering potential for selected target receptors, with binding energies of −8.035 (S-glycoprotein) and −7.685 kcal/mol (NRP1), respectively. Furthermore, computational studies on other SARS-CoV-2 strains resulting from mutations in the original wild strain (V483A, N501Y-K417N-E484K, N501Y, N439K, L452R-T478K, K417N, G476S, F456L, E484K) revealed that virus internalization activity was maintained, but with different single compound contributions

ACE and α‐glucosidase inhibitory activity of methanol extract of Alchemilla viridiflora Rothm. (Rosaceae)

Tanini, polifenolni biljni metaboliti, značajno smanjuju postprandijalnu hiperglikemiju inhibicijom α‐glukozidaze, i stoga mogu biti efikasna strategija u kontroli dijabetesa tipa 2. Takođe, dokazano je i da nespecifično inhibiraju aktivnost angiotenzin‐konvertujućeg enzima (ACE). Kako su tanini identifikovani samo u vrsti Alchemilla vulgaris L., cilj ovog istraživanja je da se odredi sadržaj tanina u do sada neistraženoj vrsti A. viridiflora Rothm. (Rosaceae), kao i inhibitorni uticaj na aktivnost ACE i α‐glukozidaze. Ukupni sadržaj tanina u metanolnom ekstraktu A. viridiflora određen je prema propisu Ph. Eur. 9.0. Suvi metanolni ekstrakt, enzimski rastvor (400 mU/ml α‐ glukozidaze u 0,1 M fosfatnom puferu) i supstrat, p‐nitrofenil α‐D‐glukopiranozid korišćeni su za kolorimetrijski test inhibitorne aktivnosti α‐glukozidaze. Kao pozitivna kontrola korišćena je akarboza. ACE inhibitorna aktivnost metanolnog ekstrakta ispitana je korišćenjem komercijalnog testa ACE Kit‐ WST (Dojindo Inc., Japan) prema uputstvu proizvođača. Procenat inhibicije enzima je izračunata IC50 vrednost, tj. procenjena koncentracija ekstrakta koja je izazvala 50% inhibicije aktivnosti enzima, koristeći linearnu regresionu analizu. IC50 vrednost metanolnog ekstrakta A. viridiflora, očitana sa dozno‐zavisne krive iznosi 2,6±0,5 μg/ml, i ekstrakt pokazuje bolju anti‐α‐glukozidaznu aktivnost od standarda akarboze (IC50=74,2±3,3 μg/ml). Takođe, ispitivani ekstrakt pokazuje doznozavisnu inhibiciju ACE pri IC50 2 μg/ml. Dobijeni rezultati su u korelaciji sa visokim sadržajem tanina u metanolnom ekstraktu A. viridiflora (3,74 %). Pokazane inhibicije angiotenzin‐konvertujućeg enzima i α‐glukozidaze čine metanolni ekstrakt vrste A. viridiflora pogodnim za dalje istraživanje u cilju pronalaženja novih prirodnih proizvoda značajnih za terapiju kardiovaskularnih bolesti i dijabetesa.Tannins, polyphenolic plant metabolites, significantly reduce postprandial hyperglycemia by inhibiting α‐glucosidase, and therefore can be an effective strategy for controlling type 2 diabetes. It has also been proven that they are non‐specific inhibitors of the activity of angiotensin‐converting enzyme (ACE). As tannins were identified only in the species Alchemilla vulgaris L., the aim of this study is to determine the content of tannins in the unexplored A. viridiflora Rothm. (Rosaceae), as well as the inhibitory effect on the activity of angiotensin‐converting enzyme and α‐glucosidase. The content of tannins in methanol extract of A. viridiflora was determined according to the Ph. Eur. 9.0. Dry methanol extract, enzyme solution (400 mU/ml of α‐ glucosidase in 0.1 M phosphate buffer) and substrate, p‐nitrophenyl α‐Dglucopyranoside were used for colorimetric α‐glucosidase inhibitory activity test. Acarbose was used as a positive control. The ACE inhibitory activity of the methanol extract was tested using the commercial ACE Kit‐WST (Dojindo Inc., Japan) according to the manufacturer's instructions. The percentage of enzyme inhibition is the calculated IC50 value, i.e. estimated concentration of the extract that caused 50% inhibition of enzyme activity using linear regression analysis. The IC50 of A. viridiflora methanol extract, read from the dose‐dependent curve, was 2.6±0.5 μg/mL, and this extract demonstrated better anti‐α‐glucosidase activity than standard acarbose (IC50=74.2±3.3 μg/mL). In addition, the examined extract shows a dose‐dependent inhibition of ACE with IC50 2 μg/mL. Obtained results were in correlation with high level of tannins in methanol extract of A. viridiflora (3.74%). The proven inhibitions of ACE and α-glucosidase make the methanol extract of A. viridiflora suitable for further scientific research in order to find a new natural product for the treatment of cardiovascular diseases and diabetes.VII Kongres farmaceuta Srbije sa međunarodnim učešćem: Zajedno stvaramo budućnost farmacije, Beograd, Srbija, 10-14. oktobar 2018.Usmeno izlaganje na skup

Essential oil composition of different parts of endemic species Seseli gracile Waldst. & Kit. (Apiaceae) from natural and cultivated conditions

The chemical composition of the essential oils of Seseli gracile Waldst. & Kit. from natural habitat (Derdap Gorge, Serbia) and from cultivated plants (Belgrade, Serbia) were characterized. The essential oils of the root, aerial parts, inflorescence and fruit were analyzed by GC/MS and GC/FID. Monoterpene hydrocarbons were the main compounds in the essential oil of aerial parts (45.2-93.0 %), inflorescences (84.1 and 90.0 %) and fruit (85.0 %). Polyacetylenes (38.8 and 87.6 %) were dominant in the essential oil of root. The cluster analysis revealed that there were significant differences in the chemical composition of the S. gracile oils at different phenological stages. On the other hand, essential oils from the aerial parts from natural and cultivated plants showed quite uniform qualitative composition. The aerial parts essential oil from natural habitat contained higher content of para-cymene (mean values 17.3 vs. 6.5 %) and lower amounts of terpinolene (mean values 23.1 vs. 49.9 %). Also polyacetylene falcarinol was present only in the aerial parts samples from natural habitat. The essential oil of inflorescences from natural habitat contained higher concentration of terpinolene, quite similar amount of para-cymene and lower content of a-pinene

Chemical characterization and ACE-inhibitory activity of acetone extract of Geranium robertianum L. flowers

Geranium robertianum L. (Geraniaceae) has been traditionally used to treat a range of ailments, including high blood pressure Recent in vitro studies have shown that some traditional usages, such as antimicrobials, were scientifically confirmed. Although the phytoconstituents of this plant species have been extensively studied, some plant parts used in traditional medicine, such as flowers, have not yet been chemically characterized. Given that the inhibition of the angiotensin converting enzyme (ACE) is one of the most significant mechanisms for decreasing blood pressure, we undertook this investigation to find scientific support for G. robertianum anti-hypertensive traditional use. Acetone was used to extract plant material since prior research revealed that it had the highest total phenol and total flavonoid levels, which are considered to be the primary sources of bioactive chemicals. The chemical composition of G. robertianum collected in R. Srpska was determined using the LC MS method, and the ACE inhibitory activity of acetone extract was measured through the enzymatically cleaved 3-hydroxybutyric acid from 3- hydryoxybutyryl-gly-gly-gly. Geraniin, a hydrolysable tannin with a Mr of 952.6, was the most abundant single chemical found in the extract. Derivatives of galic and ellagic acids, as well as flavonoids like kaempferol and quercetine, a were also present in significant amounts. G. robertianum acetonic floral extract has been shown to be an effective natural ACE inhibitor with an IC50 of 22.14 μg/mL. To the best of our knowledge, this is the first report on the chemical composition of G. robertianum floral acetonic extract and its ACE inhibitory activity.XI International Symposium of Agricultural Sciences "AgroReS 2022", 26-28. May, 2022, Trebinje, Bosnia and Herzegovin

Design, Synthesis and Pharmacological Evaluation of novel N-{4-[2-(4-aryl-piperazin-1-yl)-ethyl]-phenyl}-arylamides

5HT1A receptor targeting drugs have been used as the treatment for the many neuropsychiatric disorders, such as schizophrenia and depression. As a part of ongoing research, we designed series of new compounds that share arylpiperazine common structural motif with the 5HT1A receptor ligand aripiprazole. Receptor-ligand interactions were determined by the molecular docking simulations, revealing the positive impact of the phenyl substitution in the arylpiperazine part of the molecules. Nine selected compounds were synthesized in four reaction steps in high overall yields (59-73%). In vitro pharmacological evaluation of the synthesized compounds revealed three compounds (5b, 6b and 6c) with high 5HT1A binding affinity, comparable with aripiprazole (Ki 12.0, 4.8, 12.8, 5.6 nM, respectively). Compounds from b series, 5b and 6b, possess 2-methoxyphenyl substituents, while 6c possess 2,3-dichlorophenyl substituent in the arylpiperazine part of the molecule. The pharmacological results are therefore in accordance with the molecular docking simulations thus proving the rational design. Compounds 5c, 6b and 6c can be considered as the candidates for further evaluation as new, potential antidepressants