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.

Conformation Analysis and Stereodynamics of Symmetrically ortho-Disubstituted Carvacrol Derivatives.

The design and synthesis of analogs of natural products can be a valuable source of medicinal preparations for the pharmaceutical industry. In the present study, the structural elucidation of eleven derivatives of 2,4-dihalogeno substituted synthetic analogues of the natural compound carvacrol was carried out by means of NMR experiments, and of another thirteen by DFT calculations. By selective NOE experiments and the irradiation of CH signals of the isopropyl group, individual conformers were assigned as syn and anti. By comparing GIAO/B3LYP/6-311++G(d,p)-calculated and experimentally measured vicinal 3JCH spin-spin constants, this assignment was confirmed. An unusual relationship is reported for proton-carbon vicinal couplings: 3JCH (180°) < 3JCH (0°). The conformational mobility of carvacrols was studied by 2D EXSY spectra. The application of homonuclear decoupling technique (HOBS) to these spectra simplifies the spectra, improves resolution without reducing the sensitivity, and allows a systematic examination of the rotational barrier of all compounds via their CH signals of the isopropyl group in a wider temperature interval. The rate constants of the isopropyl rotation between syn and anti conformers were determined and the corresponding energy barriers (14-17 kcal/mol) were calculated. DFT calculations of the energy barriers in carvacrol derivatives allowed the determination of the steric origin of the restricted isopropyl rotation. The barrier height depends on the size of the 2- and 4-position substituents, and is independent of the derivatization of the OH group.

Bioisosteric ferrocenyl 1,3-thiazolidine-4-carboxylic acid derivatives: In vitro antiproliferative and antimicrobial evaluations.

To improve the antiproliferative effect of ALC67 (diastereomeric mixture of ethyl 2-phenyl-3-propioloyl-1,3-thiazolidine-4-carboxylate), its structure was modified via (i) bioisosteric substitution of the phenyl ring by the ferrocene unit and (ii) replacing the propiolamide side-chain in ACL67 with other acyl groups having differing electrophilicities. In this way, a small library of methyl N-acyl-2-ferrocenyl-1,3-thiazolidine-4-carboxylates (13 compounds in total) was created and characterized by spectral and crystallographic means. The last N-acylation step was highly diastereoselective toward the cis-diastereomer. In solution, most of the obtained compounds existed as a mixture of two rotamers and displayed a preference for the syn-orientation around the CN bond. A twisted 5T4 envelope conformation was adopted by the derivative containing the N-phenoxyacetyl group in the crystalline state. Two derivatives with chloroacetyl and bromoacetyl groups in the N-3 side chain were cytotoxic to fibroblasts and hepatocellular cancer cells in the low micromolar range (IC50(MRC5) = 9.0 and 11.8 µM, respectively, and IC50(HepG2) = 10.6 and 18.4 µM, respectively) causing an effect similar to the lead compound (IC50(HepG2) = 10.0 µM) and cisplatin (IC50(MRC5) = 4.0 µM and IC50(HepG2) = 7.7 µM). Several derivatives also manifested modest antimicrobial effects against the studied microbial strains (MICs in the range from 0.44 to 4.0 µmol/mL). Our findings demonstrated that the introduction of a ferrocene core facilitated the preparation of optically pure analogs of ALC67 and that the cytotoxicity of compounds may be enhanced by adding proper electrophilic centers to the N-acyl side-chain.

Synthesis of Small Libraries of Natural Products: Part II: Identification of a New Natural Product from the Essential Oil of Pleurospermum austriacum (L.) Hoffm. (Apiaceae).

Herein, comprehensive data of NMR, MS, IR, and gas chromatography (RI) obtained by GC-MS on commonly used capillary columns of different polarity (non-polar DB-5MS and polar HP-Innowax) of a series of esters of all constitutional isomers of hexanoic acid with a homologous series of ω-phenylalkan-1-ols (phenylmethanol, 2-phenylethanol, 3-phenylpropan-1-ol, 4-phenylbutan-1-ol, and 5-phenylpentan-1-ol) and phenol, in total 48 chemical entities, were collected. The created synthetic library allowed the identification of a new constituent of the P. austriacum essential oil (3-phenylpropyl 2-methylpentanoate). The accumulated spectral and chromatographical data, as well as the established correlation between RI values and structures of regioisomeric hexanoates, provide (phyto)chemists with a tool that will make future identification of related natural compounds a straightforward task.

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.

Analysis of Polycyclic Aromatic Hydrocarbons and Phthalate Esters in Soil and Food Grains from the Balkan Peninsula: Implication on DNA Adduct Formation by Aristolochic Acid I and Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial nephropathy affecting residents of rural farming areas in many Balkan countries. Although it is generally believed that BEN is an environmental disease caused by multiple geochemical factors with much attention on aristolochic acids (AAs), its etiology remains controversial. In this study, we tested the hypothesis that environmental contamination and subsequent food contamination by polycyclic aromatic hydrocarbons (PAHs) and phthalate esters are AA toxicity factors and important to BEN development. We identified significantly higher concentrations of phenanthrene, anthracene, diethyl phthalate (DEP), dibutyl phthalate (DBP), and benzyl butyl phthalate (BBP) in both maize and wheat grain samples collected from endemic villages than from nonendemic villages. Other PAHs and phthalate esters were also detected at higher concentrations in the soil samples from endemic villages. Subsequent genotoxicity testing of cultured human kidney cells showed an alarming phenomenon that phenanthrene, DEP, BBP, and DBP can interact synergistically with AAs to form elevated levels of AA-DNA adducts, which are associated with both the nephrotoxicity and carcinogenicity of AAs, further increasing their disease risks. This study provides direct evidence that prolonged coexposure to these environmental contaminants via dietary intake may lead to greater toxicity and accelerated development of BEN.

Synthesis and antimicrobial activity of (-)-cleistenolide and analogues.

Using the "chiral pool" approach, two modified total syntheses of the biologically active δ-lactone cleistenolide (1) have been achieved starting from d-glucose. These approaches also enabled the preparation of novel analogues and derivatives of natural product 1. The applied strategy for the synthesis of 1 involves: the initial degradation of the chiral precursor for a single C-atom, C2-fragment chain extension using Z-selective Wittig reaction, and the final δ-lactonization. All tested cleistenolide analogues displayed antimicrobial activity against a panel of nine microbial strains, most of them superseding the activity of cleistenolide itself, and, in some cases, coming close in value to the observed minimal inhibitory concentrations of chloramphenicol. Increased lipophilicity of the derivatives and the non-sterically congested conjugated lactone moiety were a prerequisite for analogues with high inhibitory activity against S. aureus and, in general, Gram-positive bacteria.

Scilla bifolia Wax as a Source of Diverse Long-Chain Resorcinols and Alkane-1,3-diols.

GC, GC/MS and NMR analyses of Scilla bifolia washings allowed for the identification of thirty-six long-chain compounds belonging to six homologous series (five of which are from the class of resorcinols, a group of biologically important phenols): 1-alkyl-3,5-dimethoxybenzenes, 5-alkyl-3-methoxy-2-methylphenols, 3-alkyl-5-methoxyphenols, 5-alkyl-2-methylresorcinols (five compounds from each of the series); 5-alkylresorcinols (six compounds) and 1,3-alkanediols (ten compounds). Many of these compounds rarely occur in Nature. Retention indices of these compounds, as well as indices of the corresponding trimethylsilyl derivatives, were reported, some of them for the first time. The exact regiochemistry was unambiguously determined by two-dimensional NMR experiments; in some cases, the complete NMR assignment was augmented by computer spin-simulation of 1 H-NMR spectra.

Very-Long-Chain Wax Constituents from Primula veris and P. acaulis: Does the Paradigm of Non-Branched vs. Branched Chain Dominance Universally Hold in all Plant Taxa?

Herein n-, iso- and anteiso-series of very-long-chained (VLC) alkanes (C21 -C35 ), fatty acid benzyl esters (FABEs; C20 -C32 ), and 2-alkanones (C23 -C35 ) were identified in the wax of Primula veris L. and P. acaulis (L.) L. (Primulaceae). For the very first time in a sample of natural origin, the presence of iso- and anteiso-VLC FABEs and 2-alkanones was unequivocally confirmed by synthetic work, derivatization, and NMR. It should be noted that the studied species produced unusually high amounts of branched wax constituents (e. g., >50 % of 2-alkanones were branched isomers). The domination of iso-isomers, probably biosynthesized from leucine-derived starters, is a unique feature in the Plant Kingdom. The plant organ distribution of these VLC compounds in P. acaulis samples (different habitats and phenological phases) pointed to their possible ecological value. This was supported by a eutectic behavior of binary blends of FABEs and alkanes, as well as by high UV-C absorption by FABEs.

Immunomodulatory Constituents of Conocephalum conicum (Snake Liverwort) and the Relationship of Isolepidozenes to Germacranes and Humulanes.

Structural elucidation of three new sesquiterpenoids, namely, (1Z,4E)-lepidoza-1(10),4-dien-14-ol (1), rel-(1(10)Z,4S,5E,7R)-germacra-1(10),6 diene-11,14-diol (2), and rel-(1(10)Z,4S,5E,7R)-humula-1(10),5-diene-7,14-diol (3), isolated from the liverwort Conocephalum conicum, was accomplished by a combination of extensive NMR experiments, 1H NMR simulation, and other means. Additionally, the change of the identity of bicyclogermacren-14-al, previously reported as a C. conicum constituent, to isolepidozen-14-al is proposed. Compounds 2 and 3 appear to be related to 1 via hydration involving a shared intermediate, a substituted cyclopropylmethyl cation, formed by a highly regio- and stereoselective protonation of 1, followed by a stereospecific fission of the three-membered ring. In other words, an isolepidozene derivative might be a branchpoint to humulanes and germacranes; this transformation could be of, up to now, unknown, biosynthetic and/or synthetic relevance. Multivariate statistical analysis of the compositional data of C. conicum extract constituents was used to probe the hypothesized biochemical relations. The immunomodulatory effect of 1-3 and conocephalenol (4) was evaluated in an in vitro model on both nonstimulated and mitogen-stimulated rat splenocytes. The compounds displayed varying degrees of cytotoxicity to nonstimulated splenocytes, whereas 2 and 3 were found to exert immunosuppressive effects on concanavalin A-stimulated splenocytes while not being cytotoxic at the same concentrations.

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.

A new longipinane ketone from Achillea abrotanoides (Vis.) Vis.: chemical transformation of the essential oil enables the identification of a minor constituent.

INTRODUCTION: Minor plant constituents are difficult to identify due to the challenging isolation and acquiring of reliable spectral data. Essential oils abound in such minor constituents that might be of high importance for their (e.g. olfactory) properties. The presence of new minor constituents is usually inferred from gas chromatography mass spectrometry (GC-MS) analyses that provide only a mass spectrum and retention data, which are insufficient to allow a positive identification. OBJECTIVE: To identify a minor unknown constituent of the essential oil of Achillea abrotanoides (Vis.) Vis. (Asteraceae). METHODOLOGY: The application of chemical transformations (oxidation and reduction) performed directly on crude essential-oil samples, followed by preparative chromatography and detailed spectral analysis, to identify a new longipinane ketone from the mentioned sample. RESULTS: GC-MS analyses of the essential oil revealed, among other constituents, the presence of a known rare longipinane alcohol (α-longipinen-7ß-ol) representing 2.5% of the total GC-peak areas, and a related unidentified oxygenated sesquiterpene (3.8%). Interpretation of their mass spectra led to an assumption that the unidentified one could represent α-longipinen-7-one. Oxidation of the entire essential-oil sample by pyridinium chlorochromate confirmed the assumed relationship among the compounds and gave a simplified enriched mixture containing the ketone (ca. 16%). A straightforward chromatographic separation of the ketone was followed by corroboration of its structure by nuclear magnetic resonance (NMR) (one- and two-dimensional), infrared (IR) and MS. CONCLUSIONS: The complementing use of chemical transformations of crude essential oils, chromatographic separations, and detailed spectral analysis could have a more general application in the identification of new natural products.

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.

Diethyl-Ether Flower Washings of Dianthus cruentus Griseb. (Caryophyllaceae): Derivatization Reactions Leading to the Identification of New Wax Constituents.

Some carnation species (Dianthus spp., Caryophyllaceae) exhibit a strong resistance to drought stress that is connected with the increased surface wax formation. Wax composition is unknown for the majority of Dianthus spp. Herein, mass spectral and gas chromatographic data, in combination with synthesis and chemical transformations (transesterification and synthesis of dimethyl disulfide adducts), enabled the identification of 151 constituents of diethyl-ether washings of fresh flowers of Dianthus cruentus Griseb. from Serbia. The flower wax contained, along with the dominant ubiquitous long-chain n-alkanes, homologous series of n- and branched (iso- and anteiso-) long-chain hexyl alkanoates/alkenoates and alkyl/alkenyl benzoates. The branching position in the mentioned hexyl esters was probed by synthesis of esters of three isomeric hexanols that were spectrally characterized (1 H- and 13 C-NMR, IR, MS). The washings also contained long-chain (Z)- and (E)-alkenes (C23 -C35 ) with several different double bond regiochemistries. Fifty-five of these constituents (eight hexyl esters, two benzoates, and forty-five alkenes) were detected for the first time in Plantae, while ten of these represent completely new compounds. The rare occurrence of these wax constituents makes them possible chemotaxonomic markers of this particular Dianthus sp.

Synthesis and Spectral Characterization of Asymmetric Azines Containing a Coumarin Moiety: The Discovery of New Antimicrobial and Antioxidant Agents.

Nine unsymmetrical azines containing a coumarin moiety were prepared by the reaction of the hydrazone of 4-hydroxy-3-acetylcoumarin with differently substituted aromatic aldehydes. The azines were fully spectrally characterized, including a complete assignment of 1 H- and 13 C-NMR resonances, and were assessed for their acute toxicities in the Artemia salina model. Their free radical scavenging activities were tested in the DPPH assay, and in vitro antimicrobial activities were determined against seven bacterial and two fungal strains. The azines containing a p-hydroxyphenyl group were shown to be the most effective antimicrobial agents, and in the case of resistant strains of Staphylococcus aureus and Acinetobacter baumannii, the activity was comparable to that of chloramphenicol. The derivative having a 3,5-dimethoxy-4-hydroxyphenyl group exhibited pronounced antioxidant power reacting rapidly and in 1 : 1 mol ratio with the DPPH radical.

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.

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.

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.

Prevalence of self-medication practice with herbal products among non-psychotic psychiatric patients from southeastern Serbia: A cross-sectional study.

The aim of this study was to evaluate the usage prevalence of herbal products (HP) and to ascertain the identity, mode and adverse effects of plant taxa used in self-medication practice for anxiety, depression and insomnia in patients with non-psychotic disorders originating from southeastern Serbia. Also, we compared HP users and non-users on the variables of socio-demographic characteristics, information source and origin of HP. The study was done by a face-to-face interview with a trained psychiatrist using a structured questionnaire administered to 136 adult patients suffering from non-psychotic mental disorders. A typical herbal-product user among non-psychotic psychiatric patients from southeastern Serbia is a middle-aged married woman, with a secondary level of education, unemployed and living in an urban area. Non-psychotic psychiatric patients, although not living predominantly in rural areas, were familiar with a variety of ethno-medicines and were often using HP primarily without the consultation of their psychiatrists/physicians. HP stated to be most frequently used for psychiatry-related symptoms included: Melissa officinalis, Mentha × piperita, Hypericum perforatum and Valeriana officinalis. The interviewees rarely stated adverse reactions related to the HP usage; however, this should not be generalized, since HP are known to vary in the content of their adverse reaction-causing constituents.