Nikola M. Stojanović

Antimicrobial Plant Metabolites: Structural Diversity and Mechanism of Action

Microbial infectious diseases continue to be one of the leading causes of morbidity and mortality. It has been estimated that microbial species comprise about 60% of the Earths biomass. This, together with the fact that their genetic, metabolic and physiological diversity is extraordinary, makes them a major threat to the health and development of populations across the world. Widespread antibiotic resistance, the emergence of new pathogens in addition to the resurgence of old ones, and the lack of effective new therapeutics exacerbate the problems. Thus, the need to discover and develop new antimicrobial agents is critical to improve mankinds future health. Plant secondary metabolites (PSMs) offer particular promise in this sense. Plant Kingdom could be considered a rich source of the most diverse structures (e.g. there are more than 12,000 known alkaloids, more than 8,000 phenolic compounds and over 25,000 different terpenoids), many of which were proven to possess strong antimicrobial properties (e.g. thymol, eurabienol, etc.). In many instances, PSMs can be easily isolated from the plant matrix, either in pure state or in the form of mixtures of chemically related compounds. What is also important is that the development of bacterial resistance toward natural plant products (that are generally regarded as eco-friendly) has been thus far documented in a very limited number of cases (e.g. for reserpine). Having all of the mentioned advantages of PSMs as potential antimicrobials in mind, a major question arises: why is it that there are still no commercially available or commonly used antibiotics of plant origin? This review tries to give a critical answer to this question by considering potential mechanisms of antimicrobial action of PSMs (inhibition of cell wall or protein synthesis, inducing leakage from the cells by tampering with the function of the membranes, interfering with intermediary metabolisms or DNA/RNA synthesis/function), as well as their physical and chemical properties (e.g. hydrophilicity/lipophilicity, chemical stability). To address the possible synergistic/antagonistic effects between PSMs and with standard antibiotics, special attention has been given to the antimicrobial activity of PSM-mixtures (e.g. essential oils, plant extracts). Moreover, possible ways of overcoming some of PSMs molecular limitations in respect to their usage as potential antibiotics were also discussed (e.g. derivatization that would enable fine tuning of certain molecular characteristics).

Toxic essential oils. Part II: Chemical, toxicological, pharmacological and microbiological profiles of Artemisia annua L. volatiles

Botanical drugs based on Artemisia annua L. (Asteraceae) are important in the treatment of malaria. Alongside with artemisinin, this aromatic species produces high and variable amounts of other chemicals that have mostly unknown biological/pharmacological activities. Herein, we have studied the toxicological/pharmacological profile of volatile constituents of a Serbian population of A. annua. Fifty-eight components were identified, among them, artemisia ketone (35.7%), α-pinene (16.5%) and 1,8-cineole (5.5%) were the most abundant ones. Significant variability of A. annua volatile profile was confirmed by means of agglomerative hierarchical cluster analysis indicating the existence of several different A. annua chemotypes. In an attempt to connect the chemical profile of A. annua oil with its biological/toxicological effects, we have evaluated in vivo and/or in vitro toxicity (including hepato- and nephrotoxicity/protection), antinociceptive, antioxidant (DPPH, ABTS and superoxide radical scavenging activity assays), enzyme inhibiting (protein kinase A and α-amylase) and antimicrobial potential of A. annua oil and of its constituents. Our results revealed that the beneficial properties of A. annua botanical drugs are not limited only to their antimalarial properties. Taking into account its relatively low toxicity, the usage of A. annua volatiles (at least of the herein studied population) does not represent a health risk.

Toxic essential oils: Anxiolytic, antinociceptive and antimicrobial properties of the yarrow Achillea umbellata Sibth. et Sm. (Asteraceae) volatiles

Many plant species are used for medicinal purposes without the knowledge of their possible toxic effect. The ethnopharmacologically renowned genus Achillea L. (Asteraceae) is even more troublesome in this respect since different taxa are believed to have the same beneficial properties as A. millefolium. According to the median lethal i.p. dose (LD(50)=853 mg/kg, mice), the volatiles of Achillea umbellata Sibth. et Sm. are more toxic than the thujone-containing essential oils (LD(50)>960 mg/kg). A GC-MS analysis of A. umbellata oil revealed the presence of a series of fragranyl esters (six new natural products). The major constituents of this oil, the rare monoterpene alcohol fragranol and fragranyl acetate, and one more ester (benzoate), as well as the oil itself, showed antianxiety, analgesic and, in some instances, paralyzing properties at 50-150 mg/kg but these are very likely sign of intoxication and not of possible beneficial effects of the plant volatiles. Testing of antimicrobial activity demonstrated that the oil possesses moderate activity against pathogenic microorganisms, but the effect of the oil differs in pro- and eukaryotic cells. According to the results obtained, fragranol may be considered as the main active principle responsible for the observed activity/toxicity.

Effects of Methyl and Isopropyl N-methylanthranilates from Choisya ternata Kunth (Rutaceae) on Experimental Anxiety and Depression in Mice

Choisya ternata Kunth (Rutaceae) is a plant species used in Mexican folk medicine for its antispasmodic and simulative properties. Recently, we identified a new alkaloid, isopropyl N‐methylanthranilate, and a related one, methyl N‐methylanthranilate, from the essential oil of this species and have proven them to possess antinociceptive activity even at 0.3 mg/kg. In the present study, anxiolytic and antidepressant effects of the two compounds have been studied in open field, horizontal wire, light/dark, forced swimming and tail suspension tests, as well as the effect on the onset and duration of diazepam‐induced sleep in BALB/c mice. The volatile alkaloids (50–200 mg/kg, administered intraperitoneally), without having a muscle relaxant effect, caused a significant increase in the time the animals spent in an unsecured and putatively dangerous area when compared with the control group but had no effect on the number of crossings between the light/dark compartments. In addition to this anxiolytic activity, a significantly antidepressant‐like effect was apparent at all tested doses, which was not due to an increase in locomotive activity. The anthranilates administered on their own did not induce sleep in mice but significantly prolonged the diazepam‐induced sleep, in a dose‐dependent way, suggesting an interaction with the gamma‐aminobutyric acid receptor complex. Copyright

Toxic essential oils. Part IV: The essential oil of Achillea falcata L. as a source of biologically/pharmacologically active trans-sabinyl esters

Herein we report on the comprehensive chemical analysis of the essential oils obtained from above- and underground parts of a previously unreported chemotype of Achillea falcata L. (Asteraceae) and, for the first time, on the biological/toxicological profile of its dominant/newly discovered volatile metabolites. Detailed spectral analyses, in combination with chemical synthesis and theoretical study, of selected constituents, enabled the identification of trans-sabinol and its esters - the formate, tiglate (new compounds), acetate, butanoate, isobutanoate, 2-methylbutanoate and 3-methylbutanoate - in both aerial and underground parts of A. falcata. Evaluation of acute toxicity in Artemia salina model, in vitro and in silico (molecular docking) evaluation of acetylcholinesterase inhibitory activity and in vivo (mice) evaluation of antinociceptive activity (hot plate, tail immersion and acetylcholine-induced abdominal writhing tests) of trans-sabinol and its esters suggested that they may interact with different targets in crustacean/mammalian organisms. Alongside moderate acute toxicity (LD50 (48 h) = 0.03-0.26 mmol/L), the tested compounds exert influence on both the peripheral and central nervous systems (in the hot plate test, trans-sabinyl tiglate, at 50 mg/kg, produced a 140% baseline increase 15 min after the treatment) and to moderately inhibit acetylcholinesterase (at the concentration of 20 µg/mL, these compounds caused a reduction of acetylcholinesterase activity up to 40%).

Methyl and isopropyl N-methylanthranilates attenuate diclofenac- and ethanol-induced gastric lesions in rats

AIMS Two natural alkaloids, methyl (M) and isopropyl (I) N-methylanthranilates, with recently demonstrated significant pharmacological activities, were assayed for their possible overall effect on intact gastric mucosa and their protective properties towards the onset of gastric lesions induced by diclofenac (a non-steroidal anti-inflammatory drug, NSAID) or ethanol. MAIN METHODS The influence of I and M on gastric mucosa integrity was assessed by oral administration in doses of 200mg/kg. The gastroprotective action of I and M in doses of 50, 100 and 200mg/kg was analyzed in the diclofenac and ethanol-induced gastric lesion models in rats. After the treatment, the stomachs of the animals were analyzed (captured by a digital camera). Ulcer scoring, morphometric and histopathological analyses of the stomachs were done. KEY FINDINGS The oral application of these compounds on their own, even in quite high doses (200mg/kg) did not induce gastric lesions. Both alkaloids exerted a very strong antiulcer activity, even in low doses (50mg/kg), by decreasing the number of lesions caused by the application of either diclofenac or ethanol, eliminating them completely or reducing them to a form of mucosal hyperemia. SIGNIFICANCE Their possible mechanism of action was discussed and due to their many positive properties including anxiolytic, antidepressant, antinociceptive, anti-inflammatory and gastroprotective activities, as well as a cheap and simple synthetic route for their preparation, methyl and isopropyl N-methylanthranilates, both alike, might represent a cost effective alternative sought for in the treatment of peptic ulcers and/or new safer NSAIDs for pain management.

The Last Decade of Antinociceptive Alkaloids: Structure, Synthesis, Mechanism of Action and Prospect

High molecular diversity of natural products (NPs) can provide a solution for many serious medicinal conditions. Frequently attributed with strong and useful biological/pharmacological properties, NPs may be directly applied in therapy, or could serve as templates for future drugs. As pain is a major symptom in many illnesses, and can significantly interfere with a persons quality of life and general functioning, pain-killing potential is certainly among the most highly valued features of any newly discovered, potentially pharmacologically useful molecule. Inspired by the fact that some of the most famous and powerful analgesic/antinociceptive agents are natural (plant) alkaloids (e.g. codeine, morphine), or are derived from them (oxycodone), herein we have tried to systematize recent findings (accumulated during the last decade) on molecular structure, mechanism of antinociceptive/analgesic action and synthesis of pain-killing alkaloids. In other words, this review tries to find out whether it is possible to give a general answer to the following questions: Which (new) structures are active? How can we obtain them? What do they do to the organism? How do they do that (structure-activity relationship)? And what can we do to them to make them better (i.e. could they be used as potential leads for new antinociceptive drugs)?

Two goitrogenic 1,3-oxazolidine-2-thione derivatives from Brassicales taxa: Challenging identification, occurrence and immunomodulatory effects

1,3-Oxazolidine-2-thione derivatives are glucosinolate-related food constituents known to impart (thyreo)toxic properties to some cruciferous vegetables. In this work, 5,5-dimethyl-1,3-oxazolidine-2-thione and (-)-(R)-5-phenyl-1,3-oxazolidine-2-thione, known goitrogens, were isolated from Draba lasiocarpa Rochel (Brassicaceae) and Reseda luteola L. (Resedaceae), respectively, and were fully spectrally characterized. Subsequently, the occurrence of the two 1,3-oxazolidine-2-thiones was verified in six additional taxa out of in total 78 screened Serbian Brassicales taxa. The stereochemistry of 5-phenyl-1,3-oxazolidine-2-thione was inferred from nuclear magnetic resonance experiments with a chiral lanthanide-shift reagent, employed in this work for the first time for this type of compounds. Unexpectedly, during gas chromatography, 5-phenyl-1,3-oxazolidine-2-thione underwent an unreported thermal core isomerization (1,3-oxazolidine-2-thione to 1,3-thiazolidine-2-one). These goitrogenic volatile glucosinolate products were tested for their effect on rat macrophage viability (three assays) and nitric oxide production. It was shown that the compounds displayed different levels of cytotoxicity. All tested compounds caused a significant lactate dehydrogenase leakage, but only (R)-5-phenyl-1,3-oxazolidine-2-thione statistically significantly reduced macrophage mitochondrial activity, whereas the racemic 5-phenyl-1,3-oxazolidine-2-thione and 5,5-dimethyl-1,3-oxazolidine-2-thione had little or no effect. Again only (R)-5-phenyl-1,3-oxazolidine-2-thione exerted nitric oxide production-inhibiting properties, suggesting the higher immunomodulatory potential of this enantiomer compared with its antipode and racemic mixture.

Immunomodulatory pinguisane-type sesquiterpenes from the liverwort Porella cordaeana (Porellaceae): the “new old” furanopinguisanol and its oxidation product exert mutually different effects on rat splenocytes

Two new natural products, α-furanopinguisanol (1) and furanopinguisanone (2), were identified in the liverwort Porella cordaeana (Porellaceae). Although α-furanopinguisanol was previously reported in the literature, herein, we present arguments that its structure was actually erroneously assigned to a different, related compound. Structure elucidation was accomplished by spectral means (various 1D and 2D NMR experiments, IR, UV, MS) and the structures corroborated by chemical transformations. The relative configuration of the compounds was additionally verified by an analysis of shift changes produced by a lanthanide shift reagent. Immunomodulatory properties of these two compounds were also investigated. Their influence on rat splenocytes (SPCs) was monitored through MTT, trypan blue and neutral red assays, microscopic investigation of cells, comet assay, and protein, RNA and DNA contents. It was shown that, in higher concentration (10−4 M), compound 1 induced a blast-like transformation of SPCs, while in lower ones (10−8 to 10−6 M) it acted as a cytotoxic agent. On the other hand, compound 2 exerted prominent cytotoxicity in all concentrations. It the light of the obtained data, a possible mechanism of action of the two compounds was discussed.

Surveillance and characterization of Candida bloodstream infections in a Serbian tertiary care hospital

INTRODUCTION Candida spp. frequently cause hospital-acquired bloodstream infections (BSI) with a high mortality rate (up to 70%). We analyzed the frequency, infection characteristics, potential predisposing factors, susceptibility to antifungal drugs, biofilm production and other virulence characteristics of Candida spp. isolates obtained from a tertiary care hospital in Niš, Serbia, during a one year period. METHODS Medical histories, characteristics of isolated strains and drug susceptibility, as well as the effect on the function of isolated macrophages and other virulence features were evaluated. The obtained results were subjected to students t-test and multivariate statistical analyzes. RESULTS Herein we report an annual incidence of 3.65 cases of C. albicans, C. lusitaniae and C. lipolytica infections per 105 population. Out of eight isolated strains, two (25%) were shown to be strong biofilm producers, one (12.5%) caused hemolysis on blood agar and in two (25%) cases macrophages were able to completely eliminate the yeast colonies. Chronic kidney disease, diabetes, malignant and other diseases were present in 37.5, 62.5, 50 and 75%, respectively, in the study group. All patients with Candida BSI received antifungal therapy (amphotericin B), however, hospital mortality was observed in 25% of patients. CONCLUSIONS Evaluation of local Candida epidemiology, antifungal susceptibility and virulence factors, as well as personalized patient risk factors are important for the surveillance of Candida BSI, especially in intensive care unit patients and may contribute to the improved options and outcome for patients with Candida BSI.