Pavle J. Randjelović

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.

Cytoprotective effect of vitamin C against gentamicin-induced acute kidney injury in rats

Since gentamicin-induced nephrotoxicity has very important clinical consequences, different potentially therapeutic approaches to prevent or attenuate it have been proposed. Accordingly, this study aimed at determining the possible protective effects of vitamin C against gentamicin-associated acute kidney injury. Experiments were done on 40 adult Wistar rats divided into four groups of 10 animals each. G-group received gentamicin (100 mg/kg) while GVC-group received the same dose of gentamicin and vitamin C (200 mg/kg) by intraperitoneal injections on a daily basis. Animals in VC-group, serving as a positive control, received only vitamin C (200 mg/kg), and those in C-group, serving as a negative control, received saline (1 ml/day), both given intraperitoneally. All groups were treated during 8 consecutive days. Quantitative evaluation of gentamicin-induced structural alterations and degree of functional alterations of kidney were performed by histopathological, morphometrical and biochemical analyses in order to determine potential beneficial effects of vitamin C co-administration with gentamicin. In G-group the proximal convoluted tubules showed cytoplasm vacuolation with dark inclusions in the epithelial cells and coagulation-type necrosis, while in GVC-group necrosis was not observed. The glomerular basement membrane was significantly thickened (p<0.05) in G-group animals than in other groups. Nuclear optical density of the tubular epithelial cells in GVC-group was significantly higher (p<0.05) compared to G-group. Blood urea and serum creatinine concentration were significantly elevated, while potassium concentration was lowered in G-group compared to other groups (p<0.01 for each). Concomitant administration of gentamicin and vitamin C resulted in a significant reduction of morphological and functional kidney alterations.

Salicylic Acid Attenuates Gentamicin-Induced Nephrotoxicity in Rats

Gentamicin (GM) is a widely used antibiotic against serious and life-threatening infections, but its usefulness is limited by the development of nephrotoxicity. The present study was designed to determine the protective effect of salicylic acid (SA) in gentamicin-induced nephrotoxicity in rats. Quantitative evaluation of gentamicin-induced structural alterations and degree of functional alterations in the kidneys were performed by histopathological and biochemical analyses in order to determine potential beneficial effects of SA coadministration with gentamicin. Gentamicin was observed to cause a severe nephrotoxicity which was evidenced by an elevation of serum urea and creatinine levels. The significant increases in malondialdehyde (MDA) levels and protein carbonyl groups indicated that GM-induced tissue injury was mediated through oxidative reactions. On the other hand, simultaneous SA administration protected kidney tissue against the oxidative damage and the nephrotoxic effect caused by GM treatment. Exposure to GM caused necrosis of tubular epithelial cells. Necrosis of tubules was found to be prevented by SA pretreatment. The results from our study indicate that SA supplement attenuates oxidative-stress associated renal injury by reducing oxygen free radicals and lipid peroxidation in gentamicin-treated rats.

Protective effect of selenium on gentamicin-induced oxidative stress and nephrotoxicity in rats

Gentamicin (GM) is a widely used antibiotic against serious, life-threatening infections, but its usefulness is limited by the development of nephrotoxicity. The present study was designed to determine the protective effect of selenium (Se) in GM-induced nephrotoxicity in rats. Experiments were done on 32 adult Wistar rats divided into four groups of 8 animals each. The GM group received gentamicin (100 mg/kg), whereas the GM+Se group received the same dose of GM and selenium (1 mg/kg) by intraperitoneal (i.p.) injections on a daily basis. Animals in the Se group, serving as a positive control, received only selenium (1 mg/kg) and the control group received saline (1 mL/day), both given i.p. All groups were treated during 8 consecutive days. Quantitative evaluation of GM-induced structural alterations and degree of functional alterations in the kidneys were performed by histopathological and biochemical analyses in order to determine potential beneficial effects of selenium coadministration with GM. GM was observed to cause a severe nephrotoxicity, which was evidenced by an elevation of serum urea and creatinine levels. The significant increases in malondialdehyde levels and protein carbonyl groups indicated that GM-induced tissue injury was mediated through oxidative reactions. On the other hand, simultaneous selenium administration protected kidney tissue against oxidative damage and the nephrotoxic effect caused by GM treatment. Exposure to GM caused necrosis of tubular epithelial cells. Necrosis of tubules was found to be prevented by selenium pretreatment. The results from our study indicate that selenium supplementation attenuates oxidative-stress–associated renal injury by reducing oxygen free radicals and lipid peroxidation in GM-treated rats.

Protective Effects of Pentoxifylline Treatment on Gentamicin-Induced Nephrotoxicity in Rats

Gentamicin (GM) is a widely used antibiotic against serious and life-threatening infections, but its usefulness is limited by the development of nephrotoxicity. Thus, the present study was undertaken to determine if pentoxifylline could protect the kidney in this experimental model. Thirty male Wistar rats were used. The animals were divided into three groups, each with 10 animals. The GM group of animals was treated daily with gentamicin in a dose of 100 mg/kg for eight days. The GMP group of animals was treated daily with pentoxifylline in a dose of 45 mg/kg and the same dose of gentamicin as the GM group for eight days. The control group received 1 mL/day saline intraperitoneally. For histological analysis, 5 μm-thick sections were stained with hematoxylin and eosin (HE), periodic acid Schiff (PAS), and Jones methenamine silver. The morphometric parameters included were glomerular area, major and minor axis, perimeter, diameter, roundness, and mean optical density. Biochemical analyses were used to determine concentrations of blood urea, serum creatinine, sodium, and potassium. In the GM group of rats, glomerular basement membrane was diffusely and unequally thickened with polymorphonuclear leukocyte infiltration, and coagulation-type necrosis and vacuolization of cytoplasm of proximal tubules epithelial cells were observed. In the GMP group of rats, glomeruli were slightly enlarged with thickened basement membrane in some segments but without coagulation-type necrosis of proximal tubules epithelial cells. Blood urea and serum creatinine concentration in the GM group were significantly elevated in comparison with the GMP group, while the potassium level was decreased. The present study indicated that pentoxifylline could provide a marked protective effect against gentamicin-induced acute renal failure, most likely mediated by vascular decongestion.

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.