Marrubiin Inhibits Peritoneal Inflammatory Response Induced by Carrageenan Application in C57 Mice.

Marrubiin is a diterpene with a long history of a wide range of biological activities. In this study, the anti-inflammatory effects of marrubiin were investigated using several in vitro and in vivo assays. Marrubiin inhibited carrageenan-induced peritoneal inflammation by preventing inflammatory cell infiltration and peritoneal mast cell degranulation. The anti-inflammatory activity was further demonstrated by monitoring a set of biochemical parameters, showing that the peritoneal fluid of animals treated with marrubiin had lower levels of proteins and lower myeloperoxidase activity compared with the fluid of animals that were not treated. Marrubiin exerted the most pronounced cytotoxic activity towards peripheral mononuclear cells, being the main contributors to peritoneal inflammation. Additionally, a moderate lipoxygenase inhibition activity of marrubiin was observed.

Oregano (Origanum vulgare) Essential Oil and Its Constituents Prevent Rat Kidney Tissue Injury and Inflammation Induced by a High Dose of L-Arginine.

This study aimed to evaluate the protective action of oregano (Origanum vulgare) essential oil and its monoterpene constituents (thymol and carvacrol) in L-arginine-induced kidney damage by studying inflammatory and tissue damage parameters. The determination of biochemical markers that reflect kidney function, i.e., serum levels of urea and creatinine, tissue levels of neutrophil-gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1), as well as a panel of oxidative-stress-related and inflammatory biomarkers, was performed. Furthermore, histopathological and immunohistochemical analyses of kidneys obtained from different experimental groups were conducted. Pre-treatment with the investigated compounds prevented an L-arginine-induced increase in serum and tissue kidney damage markers and, additionally, decreased the levels of inflammation-related parameters (TNF-α and nitric oxide concentrations and myeloperoxidase activity). Micromorphological kidney tissue changes correlate with the alterations observed in the biochemical parameters, as well as the expression of CD95 in tubule cells and CD68 in inflammatory infiltrate cells. The present results revealed that oregano essential oil, thymol, and carvacrol exert nephroprotective activity, which could be, to a great extent, associated with their anti-inflammatory, antiradical scavenging, and antiapoptotic action and, above all, due to their ability to lessen the disturbances arising from acute pancreatic damage. Further in-depth studies are needed in order to provide more detailed explanations of the observed activities.

Melatonin treatment prevents carbon-tetrachloride induced rat brain injury.

Introduction: Herein the neuroprotective properties of melatonin, a highly effective antioxidant, administered in a single dose 50 mg/kg intraperitoneally, were investigated in the brain tissue of Wistar rats acutely exposed to the toxin carbon-tetrachloride (1 mL/kg, intraperitoneally). Methods: To assess the degree of whole encephalic mass damage, biochemical parameters related to lipid and protein oxidation, antioxidant enzymes (catalase and superoxide dismutase), glutathione and inducible nitric oxide/arginase pathways were determined. Results: The results showed that carbon-tetrachloride impaired the function of antioxidant enzymes (reduced catalase and superoxide dismutase activities) and reduced glutathione-metabolizing enzymes (reduced glutathione, glutathione S-transferase and peroxidase activity). Furthermore, carbon-tetrachloride increased lipid peroxidation and protein oxidative damage in the brain tissue, as well as myeloperoxidase and inducible nitric oxide synthase content/activities. Conclusions: The application of a single dose of melatonin post intoxication has been able to reverse the disturbance in the function of antioxidant enzymes and alleviate the tissue damage caused by oxidative stress, indicating that melatonin could be a potential therapeutic agent in oxidative-damage related neurodegenerative disorders.

How do different bile acid derivatives affect rat macrophage function - Friends or foes?

Due to an increased need for new immunomodulatory agents, many previously known molecules have been structurally modified in order to obtain new drugs, preserving at the same time some of the benevolent characteristics of the parent molecule. This study aimed to evaluate the immunomodulatory potential of a selected library of bile acid derivatives (BAD) using a broad spectrum of assays, evaluating rat peritoneal macrophages viability, cell membrane damage, lysosomal and adhesion function, and nitric oxide and cytokine production as a response to lipopolysaccharide stimulation. Also, in silico studies on two bile acid-activated receptors were conducted and the results were related to the observed in vitro effects. All tested BAD exerted significant toxicity in concentrations higher than 10 µM, which was determined based on mitochondria and cell membrane damage in a panel of assays. On the other hand, at lower concentrations, the tested BAD proved to be immunomodulatory since they affected lysosomal function, cell adhesion capacities and the ability to produce inflammatory cytokines in response to a stimulus. One of the compounds proved to exhibit significant toxicity toward macrophages, but also caused a concentration-dependent decrease in nitric oxide levels and was identified as a potential farnesoid X receptor agonist.

The potential of lemon balm (Melissa officinalis L.) essential oil as an anti-anxiety agent - is the citronellal the activity carrier?

ETHNOPHARMACOLOGICAL RELEVANCE: Among the fewest drugs discovered are those belonging to the class of anxiolytics. Although some drug targets for anxiety disorders are established, it is hard to modify and selectively choose the active principle for those targets. Thus, the ethnomedical approach to treating anxiety disorders remains one of the most prevalent ways for (self)managing the symptoms. Melissa officinalis L. (lemon balm) has been extensively used as an ethnomedicinal remedy for the treatment of different psyche-related symptoms, especially dose related to restlessness. AIM OF THE STUDY: This work aimed to evaluate the anxiolytic activity, in several in vivo models, of the essential oil extracted from Melissa officinalis (MO) and its main constituent citronellal, a widespread plant utilized for managing anxiety. MATERIALS AND METHODS: In the present study several animal models were used to assess MO anxiolytic potential in mice. The effect of the MO essential oil applied in doses ranging from 12.5 to 100 mg/kg was estimated in light/dark, hole board, and marble burying tests. In parallel doses of citronellal corresponding to the ones in the MO essential oil were applied to animals to determine if this is the activity carrier. RESULTS: The results indicate that the MO essential oil exerts anxiolytic potential in all three experimental settings by significantly altering the traced parameters. The effects of citronellal are somewhat inconclusive and should not be interpreted only as anxiolytic but rather as a combination of anti-anxiety and motor-inhibiting effects. CONCLUSIONS: In conclusion, we could say that the results of the present study provide a base for future mechanistic studies that would evaluate the activity of M. officinalis essential oil on various neurotransmitter systems involved in the generation, propagation, and maintenance of anxiety.

Pulicaria dysenterica (L.) Bernh.-Rightfully Earned Name? Identification and Biological Activity of New 3-Methoxycuminyl Esters from P. dysenterica Essential Oil.

Motivated by the ethnopharmacological use of Pulicaria dysenterica, in the present study, the antimicrobial potential of the extracted essential oil was investigated against a panel of eighteen microorganism strains. Additionally, anti-acetylcholinesterase and antispasmodic (isolated rat distal colon) activities, general acute toxicity (Artemia salina model), and immunomodulatory properties (cytotoxicity on isolated mouse macrophages) were studied. Detailed analyses of the essential oil led to the identification of 3-methoxycuminyl 2-methylbutanoate (a new natural product) and 3-methoxycuminyl 3-methylbutanoate (a rare natural product). The obtained esters and intermediates in the synthesis of the starting alcohol (3-methoxycuminol) were subjected to a battery of 1D- and 2D-NMR experiments. The synthesized esters were additionally characterized by GC-MS, IR, and UV-Vis. The synthesized compounds (ten in total) were biologically tested in the same way as the extracted P. dysenterica essential oil. The obtained low acute toxicity and promising antimicrobial potential suggest that the P. dysenterica essential oil might partially explain the ethnopharmacological application of P. dysenterica plant material for the treatment of gastrointestinal infections.

Lemon balm (Melissa officinalis L.) essential oil and citronellal modulate anxiety-related symptoms - In vitro and in vivo studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Besides psyche-related symptoms, patients with anxiety disorders can have a large number of somatic symptoms as well. Although the treatment of these disorders is mainly focused on resolving their mental component, one cannot neglect the need for the treatment of accompanying somatic symptoms. Melissa officinalis L. (lemon balm), in various formulations, has been extensively used as an ethnomedicinal remedy for the treatment of different psyche-related symptoms, and its use is considered relatively safe. AIM OF THE STUDY: In the present study, the activity of M. officinalis (MO) essential oil was evaluated in several in vitro and in vivo models mimicking or involving anxiety-related somatic symptoms. MATERIALS AND METHODS: To address the effect of MO essential oil on the gastrointestinal and heart-related symptoms accompanying anxiety disorders, in vitro models were utilized that follow the function of the isolated mouse ileum and atria tissues, respectively, after exposure to MO essential oil. Effects of MO essential oil on BALB/c mice motor activity was estimated using the open field, rota-rod, and horizontal wire tests. Additionally, the essential oil was assayed for its potential in inhibiting acetylcholinesterase activity. RESULTS: The performance of mice treated with 25 mg/kg of the oil showed a statistically significant decrease in the motor impairment arising from acute anxiety (open field test), while there was a prolonged latency and a reduction of the frequency of falling from a rotating rod and/or a horizontal wire (signs of muscle weakness/spasms). Concentrations of the essential oil higher than 1 µg/mL were found to inhibit both spontaneous and induced ileum contractions. Moreover, the essential oil and citronellal were found to decrease isolated mouse atria contraction frequency, as well as contraction force. However, the oil was found to be a very weak acetylcholinesterase inhibitor. CONCLUSION: The modulation of anxiety-related symptoms by the oil was found not to be mediated through the inhibition of the acetylcholinesterase, nonetheless, the mechanistic studies involving the ileum and cardiac tissues, revealed that the activity of MO and citronellal might be related to the modification of either voltage-gated Ca2+ channels or muscarinic receptors. Mice locomotion, balance, and muscle strength were not impacted by the essential oil; however, its main constituent, citronellal, was found to exert a certain degree of muscle function inhibition. All these results suggest that the activity of MO essential oil arises from synergistic and/or antagonistic interactions of its constituents, and is not completely dependent on the oil's main constituent.

Methyl and Isopropyl N-Methylanthranilates Affect Primary Macrophage Function - An Insight into the Possible Immunomodulatory Mode of Action.

To complement the knowledge on the anti-inflammatory activity of methyl and isopropyl N-methylanthranilates, two natural products with panacea-like properties, we investigated their effects on thioglycolate-elicited macrophages by evaluating macrophage ability to metabolize MTT, macrophage membrane function, and macrophage myeloperoxidase and phagocytic activities. Moreover, two additional aspects of the inflammatory response of these compounds, their inhibitory activity on xanthine oxidase and catalase, were studied. It was found that these two compounds regulate elicited macrophage functions, most probably by interfering with the function of cell membranes and changing the reducing cellular capacity or enzyme activity of macrophages. Nonetheless, no significant inhibitory action either towards xanthine oxidase or catalase was found, suggesting that the inhibition of these enzymes is not involved in the anti-inflammatory mode of action of these two esters.

Linking the antimicrobial and anti-inflammatory effects of immortelle essential oil with its chemical composition - The interplay between the major and minor constituents.

Immortelle (Helichrysum italicum, Asteraceae) essential oil has been widely used in alternative medicine to accelerate wound healing, as well as in cosmetic products to stimulate skin regeneration and to reduce the appearance of wrinkles. It is also considered a natural and safe culinary spice that could also be applied in the food industry as a preservative in the future. The therapeutic efficacy of this oil changes with the natural variability of the composition. Herein we tested and mutually compared the antimicrobial and anti-inflammatory activities of four commercial immortelle oils differing in the relative amounts of marker compounds, i.e. neryl esters, α-pinene, γ- and ar-curcumenes, and ß-diketones. The anti-inflammatory effect of selected chromatographic fractions, enriched in the aforementioned constituents, was evaluated by studying toxicity toward rat peritoneal macrophages, their nitric oxide production, myeloperoxidase, and arginase activities. Subsequently, the compositional and activity data were subjected to a multivariate statistical treatment to reveal the possible correlation(s) between the percentage of essential-oil constituents and the observed activities. The obtained results imply that immortelle oil efficiency as an antimicrobial and/or anti-inflammatory agent is most plausibly a result of a synergistic action between its constituents, and/or, rather unexpectedly, the presence of some minor constituents.

An Impact of Psychological Stress on the Interplay between Salivary Oxidative Stress and the Classic Psychological Stress-Related Parameters.

Both oxidative and psychological (mental) stress are the likely culprits for several acute and chronic health disturbances, and adequate tests mimicking that are needed. Herein, in controlled laboratory surroundings, a PEBL (Psychology Experiment Building Language) test battery was used to evoke stress-related biological responses followed by tracking changes in saliva parameters. The study objectives were to determine the impact of psychological stress on selected salivatory parameters and to assess the correlation between the determined oxidative and stress parameters. The study was conducted on 36 healthy young subjects, mainly females (n = 24). Before and following the completion of a battery of four PEBL tests, subjects' saliva samples were collected. Stress-evoking changes in total antioxidant capacity and nitrite/nitrate levels, as oxidative stress parameters, and cortisol and immunoglobulin A (IgA), as parameters of psychological stress, were established and mutually correlated by comparing the values of the evaluated parameters pre- and post-PEBL test. The results showed that there is no change in the total salivary antioxidant capacity (p > 0.05); however, there was a significant increase in nitrites/nitrates levels after the PEBL test (p = 0.007). On the other hand, the determined cortisol levels after the test battery were found to be statistically significantly increased (p = 0.025) when compared to the values obtained before the test, while the levels of IgA were found to be statistically significantly decreased (p < 0.001). The only statistically significant correlation between the changes in the studied parameters was found to be the one between cortisol and IgA levels (Spearman's Rö = -0.4). These results suggest that the short-term stress induced by the PEBL test does evoke changes in the salivary mental stress-related parameters (an increase in cortisol and nitrite/nitrate levels, and a decrease in IgA), but not in the total antioxidant capacity. They also indicate that the constructed PEBL four-test battery might represent an adequate laboratory stress-inducing paradigm.

Choosing the Right Essential Oil for a Mouthwash: Chemical, Antimicrobial and Cytotoxic Studies.

Thirteen commercial essential oils were assessed for their possible inclusion in a mouthwash formulation based on their inhibitory effect against potentially pathogenic anaerobic oral bacterial isolates from subgingival plaque, and their cytotoxicity towards gingival cells. The essential oils, originating from species belonging to seven major aromatic plant families, were chosen to provide the necessary diversity in chemical composition that was analyzed in detail by GC and GC/MS. Multivariate statistical analysis, performed using the in vitro microbiological/toxicological assays and compositional data, revealed that the major components of the essential oils were probably not the main carriers of the activities observed. A formulation of 'designer' mouthwashes is proposed based on the selective action of certain essential oils towards specific bacterial isolates (e. g., Citrus bergamia vs. Parvimonas micra), and non-toxicity to gingival cells at antimicrobially active concentrations.

Anxiolytic-Like Action of Selected 4-(Alkylamino)-3-nitrocoumarin Derivatives in BALB/c Mice.

In this work, we explored the possible polypharmacological potential of the already established antimicrobials against gastrointestinal pathogens, 4-(alkylamino)-3-nitrocoumarins, as antianxiety agents, using a battery of in vivo experiments. Three chosen coumarin derivatives, differing in the substituent (sec-butylamino, hexadecylamino, or benzylamino) at position 4, at the doses of 25, 50 and 100 mg kg-1 , were evaluated in light/dark, open-field, horizontal wire and diazepam-induced sleep models using male BALB/c mice. Depending on the applied dose, all three tested coumarins displayed a noteworthy anxiolytic-like effect. 4-(sec-Butylamino)-3-nitro-2H-chromen-2-one and 4-(hexadecylamino)-3-nitro-2H-chromen-2-one could be recognized as true anxiolytics in the lowest applied dose, based on three tests, without exerting any sedative effects. Thus, the 3-nitrocoumarin core deserves further chemical diversity exploration in the 'antianxiety' direction.

Toxic essential oils, part VI: Acute oral toxicity of lemon balm (Melissa officinalis L.) essential oil in BALB/c mice.

Despite being renowned for its volatiles, the data on the toxicity of the essential oil of lemon balm (Melissa officinalis L., Lamiaceae) is rather limited compared to its solvent/water-soluble extractibles. In this study, the aerial parts essential oil of M. officinalis, with over 130 constituents identified herein, 26 of which detected for the first time, was investigated for acute oral toxicity in BALB/c mice. The oil, composed of predominantly monoterpene aldehydes, citronellal (21.2-21.8%), neral (17.8-18.4%), and geranial (22.9-23.5%), which were assayed in parallel with the oil in some tests, induced significant changes in animal behavior, as well as altered biochemical parameters reflecting liver and kidney functions. Different pathological changes in the stomach, duodenum, liver, and kidneys were detected when the oil was administered in doses higher than 1 g kg-1. A depletion in the liver/kidney antioxidant capacities and an increased rate of lipid peroxidation was noted for animals treated with lemon balm oil. The calculated value of the oral LD50 in BALB/c mice (2.57 g kg-1) infers that the essential oil is only moderately toxic.

The Quantitative ER Immunohistochemical Analysis in Breast Cancer: Detecting the 3 + 0, 4 + 0, and 5 + 0 Allred Score Cases.

The currently used immunohistochemical approach in determining the estrogen receptor (ER) positivity of breast cancers (BCs) is inherently subjective and additionally limited by its semi-quantitative nature. The application of software in the analysis of digitized slide images may overcome some of these limitations. However, the utilization of such an approach requires that the entire staining procedure is standardized. BACKGROUND AND OBJECTIVES: We aimed to establish a procedure for the photometric and morphometric analysis of BC immunohistochemical parameters that can possibly be used for a diagnostic purpose that is in line with the current semi-quantitative scoring system. MATERIALS AND METHODS: Semi-quantitative analysis of ER-stained tissue sections was performed following the Allred scoring system guidelines. The quantitative analysis was performed in ImageJ software after color deconvolution. The quantitative analysis of 66 cases of invasive lobular BC included: Percent of ER-positive cells, average nuclear coloration intensity, and the quantitative ER score. The percent of ER-positive tumor cells was counted using a standard grid overlay, while optical density (0.0-1.0) was measured within each nucleus at the grid points. RESULTS: A statistical analysis revealed a significant positive correlation (r = 0.886, p < 0.001) between the subjective semi-quantitative and quantitative ER scores, with a large effect size (d = 3.8215). We observed strong statistically significant correlations between individual parameters of the total ER score, percentage of ER-positive nuclei, and color intensity, obtained by the two independent methods. CONCLUSIONS: Additionally, besides excluding subjectivity, the up to now unreported cases of 3 + 0, 4 + 0, and 5 + 0 Allred scores were detected only by the application of the proposed quantitative approach.

Structural Elucidation of Presilphiperfolane-7α,8α-diol, a Bioactive Sesquiterpenoid from Pulicaria vulgaris: A Combined Approach of Solvent-Induced Chemical Shifts, GIAO Calculation of Chemical Shifts, and Full Spin Analysis.

Structural elucidation of a new triquinane sesquiterpenoid, presilphiperfolane-7α,8α-diol, 1a, isolated from Pulicaria vulgaris, was accomplished by combining solvent-induced removal of chemical shift degeneracy and computational (DFT-GIAO) prediction of NMR spectra with the analysis of 1H NMR splitting patterns. In addition to extensive NMR experiments (in 10 different solvents), MS, and FTIR, the identity of 1a was also confirmed by chemical transformations. The applied approach can facilitate structural elucidation of organic molecules and decrease the probability of an erroneous identification, permitting an unambiguous stereochemical elucidation and full NMR assignment. The pharmacological/toxicological profile of 1a was evaluated in several different models.

Differences in Angular Photogrammetric Soft-Tissue Facial Characteristics among Parents and Their Offspring.

Background and objectives: The objective of this study was to determine if the angular photogrammetric analysis of soft-tissue characteristics can determine similarities between parents and their offspring in the Serbian population. Materials and Methods: A total of 15 families (52 participants) met the participation criteria of this study and their facial profile images were analyzed using the ImageJ software. Subjects were divided into groups of mothers and fathers and four groups of children (divided according to their age and gender). In total, twelve angular measurements were made on the standardized digital images of the profiles of the participants and the obtained data were compared using one-way ANOVA. Results: The obtained results showed that there were statistically significant differences in the values of the nasal and cervicomental angles, as well as the angle of the total facial convexity, between the group of fathers, on one side, and groups of male/female children, on the other. Conclusions: This work represents the first photogrammetric analysis of facial soft-tissue characteristics of children and adults in the Serbian population. The data suggest that there are much more similarities between the facial soft-tissue angles of fathers and their male offspring. Furthermore, mothers tend to have statistically insignificant differences in angle sizes, compared to both male and female offspring.

Distribution of methyl and isopropyl N-methylanthranilates and their metabolites in organs of rats treated with these two essential-oil constituents.

Two volatile alkaloids, methyl (MMA) and isopropyl N-methylanthranilates (IMA), identified in the essential oil of Choisya ternata Kunth (Rutaceae), have been proven to possess polypharmacological properties (antinociceptive, anti-inflammatory, gastro-, hepato-, nephroprotective activities, anxiolytic and antidepressant properties, and likewise an effect on diazepam-induced sleep). In the continuation of our investigation of their urinary-metabolite profiles, we performed GC-MS analyses of the diethyl-ether extracts of selected tissues (liver, kidneys, heart, brain, lungs, quadriceps femoris muscle, and spleen) of rats intraperitoneally treated with MMA or IMA (2 g kg-1). Organ-metabolite profiles of MMA and IMA were qualitatively mutually analogous (varying only in the alcohol moiety of the metabolites), and generally analogous to their urinary-metabolite profiles. The greatest diversity and the highest overall amount of anthranilate metabolites was found in the hepatic tissue. The principal anthranilate-related compounds in the organs of rats treated with MMA, among 12 detected, were the products of ester hydrolysis, N-methylanthranilic and anthranilic acids. In the tissues of IMA-treated rats, among 16 compounds, the most abundant ones were the unmetabolized IMA and N-methylanthranilic acid. A collection of the compositional data regarding the anthranilate-related metabolites was statistically treated by multivariate statistical analysis that provided a better insight into the possible biotransformation pathways.

Prenylated ß-diketones, two new additions to the family of biologically active Hypericum perforatum L. (Hypericaceae) secondary metabolites.

Two novel ß-diketones, 2,6,9-trimethyl-8-decene-3,5-dione (A) and 3,7,10-trimethyl-9-undecene-4,6-dione (B), were identified from the renowned medicinal plant Hypericum perforatum L. The structures of ß-diketones A and B were corroborated by syntheses (4 steps starting from methyl acetoacetate, overall yields 30% and 23%, respectively). In solution, these ß-diketones predominantly exist as two rapidly interconverting ß-keto-enol tautomers. The structures of A and B show some common fragments with the molecules of hyperforin and adhyperforin, respectively, the acknowledged multi-target secondary metabolites from St. John's wort. It is therefore not surprising that A displayed a noteworthy biological activity profile as well (including brine shrimp toxicity, antinociceptive, antidepressant and acetylcholinesterase inhibitory activity). ß-Diketone A manifested the most outstanding potency as an acetylcholinesterase inhibitor with IC50 value of 1.51 µM pointing again to the ß-keto-enol moiety as a promising lead structure for the development of drugs that could lessen symptoms of Alzheimer's disease (such as dementia, depression and pain).

An epidemiological study on herbal product self-medication practice among psychotic outpatients from Serbia: A cross-sectional study.

Utilization of herbal products (HPs) is a common practice in the traditional medicine of people from southeastern Serbia. In this study, we focused on the usage of HPs as a self-medication practice among patients diagnosed with a mental illness, by aiming to ascertain the usage prevalence, the identity of the main plant taxa utilized, their formulations and target symptoms. This was accomplished through a cross-sectional study of psychiatric outpatients, conducted in the Clinic for Mental Health Protection in Nis, and which included a questionnaire on HP utilization and a non-structured psychiatric interview. Typically, single, middle-aged males, with a secondary education degree, utilized Matricaria chamomilla and/or Melissa officinalis in a form of an infusion (tea) for relieving anxiety and psychotic symptoms. In some cases, adverse effects were noted when HPs were used in combination with prescribed psychotropic medications. Our and previous results urge a thorough evaluation of possible benefits and/or harmful interactions of HP with standard medication in the treatment of psychiatric patients.