Metabolic Composition of Methanolic Extract of the Balkan Endemic Species Micromeria frivaldszkyana (Degen) Velen and Its Anti-Inflammatory Effect on Male Wistar Rats.

Extracts from medicinal plants are widely used in the treatment and prevention of different diseases. Micromeria frivaldszkyana is a Balkan endemic species with reported antioxidant and antimicrobial characteristics; however, its phytochemical composition is not well defined. Here, we examined the metabolome of M. frivaldszkyana by chromatography-mass spectrometry (GC-MS), ultra-performance liquid chromatography-mass spectrometry (UPLC-MS-MS), and inductively coupled plasma mass spectrometry (ICP-MS). Amino acids, organic acids, sugars, and sugar alcohols were the primary metabolites with the highest levels in the plant extract. Detailed analysis of the sugar content identified high levels of sucrose, glucose, mannose, and fructose. Lipids are primary plant metabolites, and the analysis revealed triacylglycerols as the most abundant lipid group. Potassium (K), magnesium (Mg), zinc (Zn), and calcium (Ca) were the elements with the highest content. The results showed linarin, 3-caffeoil-quinic acid, and rosmarinic acid, as well as a number of polyphenols, as the most abundant secondary metabolites. Among the flavonoids and polyphenols with a high presence were eupatorin, kaempferol, and apigenin-compounds widely known for their bioactive properties. Further, the acute toxicity and potential anti-inflammatory activity of the methanolic extract were evaluated in Wistar rats. No toxic effects were registered after a single oral application of the extract in doses of between 200 and 5000 mg/kg bw. A fourteen-day pre-treatment with methanolic extract of M. frivaldszkyana in doses of 250, 400, and 500 mg/kg bw induced anti-inflammatory activity in the 1st, 2nd, and 3rd hours after carrageenan injection in a model of rat paw edema. This effect was also present in the 4th hour only in the group treated with a dose of 500 mg/kg. In conclusion, M. frivaldszkyana extract is particularly rich in linarin, rosmarinic acid, and flavonoids (eupatorin, kaempferol, and apigenin). Its methanolic extract induced no toxicity in male Wistar rats after oral application in doses of up to 5000 mg/kg bw. Additionally, treatment with the methanolic extract for 14 days revealed anti-inflammatory potential in a model of rat paw edema on the 1st, 2nd, and 3rd hours after the carrageenan injection. These results show the anti-inflammatory potential of the plant, which might be considered for further exploration and eventual application as a phytotherapeutic agent.

Fucoidan from Ericaria crinita Alleviates Inflammation in Rat Paw Edema, Downregulates Pro-Inflammatory Cytokine Levels, and Shows Antioxidant Activity.

Fucoidans are sulfated polysaccharides detected mainly in the cell walls of brown seaweeds. Here, we examined the effects of single doses of fucoidan derived from Ericaria crinita (formerly Cystoseira crinita) on carrageenan-induced paw inflammation in rats. The serum levels of TNF-α, IL-1ß, IL-6, and IL-10 of rats with LPS-induced systemic inflammation after 14 days of treatment were also evaluated. Subchronic treatment with fucoidan from E. crinita attenuated the inflammation during the late phase of the degraded carrageenan-induced paw edema (3rd to 5th hour after carrageenan injection) with peak activity at the 3rd hour after the application. Both doses of fucoidan from E. crinita (25 and 50 mg/kg bw) significantly decreased the levels of all tested pro-inflammatory cytokines (IL-1ß, TNF-α, and IL-6) in the serum of rats with a model of system inflammation but had no effect on the anti-inflammatory cytokine IL-10. The results showed that the repeated application of fucoidan has a more prominent effect on the levels of some pro-inflammatory cytokines in serum in comparison to a single dose of the sulfated polysaccharide. This reveals the potential of E. crinita fucoidan as an anti-inflammatory agent. Furthermore, E. crinita fucoidan exhibited in vitro antioxidant capacity, determined by 2,2-diphenyl-1-picryl-hydrazyl radical scavenging and ferric reducing antioxidant power assays as follows: IC50 = 412 µg/mL and 118.72 µM Trolox equivalent/g, respectively.

Extraction, Structural Characterization, and In Vivo Anti-Inflammatory Effect of Alginate from Cystoseira crinita (Desf.) Borry Harvested in the Bulgarian Black Sea.

The aim of this study was to identify the chemical composition and sequential structure of alginate isolated from C. crinita harvested in the Bulgarian Black Sea, as well as its effects in histamine-induced paw inflammation in rats. The serum levels of TNF-α, IL-1ß, IL-6, and IL-10 in rats with systemic inflammation, and the levels of TNF-α in a model of acute peritonitis in rats were also investigated. The structural characterization of the polysaccharide was obtained by FTIR, SEC-MALS, and 1H NMR. The extracted alginate had an M/G ratio of 1.018, a molecular weight of 7.31 × 104 g/mol, and a polydispersity index of 1.38. C. crinita alginate in doses of 25 and 100 mg/kg showed well-defined anti-inflammatory activity in the model of paw edema. A significant decrease in serum levels of IL-1ß was observed only in animals treated with C. crinita alginate in a dose of 25 mg/kg bw. The concentrations of TNF-α and IL-6 in serum were significantly reduced in rats treated with both doses of the polysaccharide, but no statistical significance was observed in the levels of the anti-inflammatory cytokine IL-10. A single dose of alginate did not significantly alter the levels of the pro-inflammatory cytokine TNF-α in the peritoneal fluid of rats with a model of peritonitis.

Structural Characterization and In Vivo Anti-Inflammatory Activity of Fucoidan from Cystoseira crinita (Desf.) Borry.

The aim of this study was to evaluate the effects of fucoidan isolated from C. crinita on histamine-induced paw inflammation in rats, and on the serum levels of TNF-α, IL-1ß, IL-6, and IL-10 in rats during systemic inflammation response. The levels of TNF-α in a model of acute peritonitis in rats were also investigated. The isolated crude fucoidan was identified as a sulfated xylogalactofucan with high, medium, and low molecular weight fractions and a content of fucose of 39.74%, xylose of 20.75%, and galactose of 15.51%. Fucoidan from C. crinita showed better anti-inflammatory effects in the rat paw edema model, and this effect was present during all stages of the experiment. When compared to controls, a commercial fucoidan from F. vesiculosus, the results also displayed anti-inflammatory activity on the 60th, 90th, and 120th minute of the experiment. A significant decrease in serum levels of IL-1ß in rats treated with both doses of C. crinita fucoidan was observed in comparison to controls, whereas TNF-α concentrations were reduced only in the group treated with fucoidan from C. crinita at the dose of 25 mg/kg bw. In the model of carrageenan-induced peritonitis, we observed a tendency of decrease in the levels of the pro-inflammatory cytokine TNF-α in peritoneal fluid after a single dose of C. crinita fucoidan, but this did not reach the statistical significance margin. Single doses of C. crinita fucoidan did not alter serum levels of the anti-inflammatory cytokine IL-10 in animals with lipopolysaccharide-induced systemic inflammation.

Immunomodulatory and Anti-Inflammatory Effects of Fucoidan: A Review.

Inflammation is the initial response of the immune system to potentially harmful stimuli (e.g., injury, stress, and infections). The process involves activation of macrophages and neutrophils, which produce mediators, such as nitric oxide (NO), prostaglandin E2 (PGE2), pro-inflammatory and anti-inflammatory cytokines. The pro-inflammatory cytokines interleukin-1ß (IL-1ß), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) are considered as biomarkers of inflammation. Even though it occurs as a physiological defense mechanism, its involvement in the pathogenesis of various diseases is reported. Rheumatoid arthritis, inflammatory bowel disease, Alzheimer's disease, and cardiovascular diseases are only a part of the diseases, in which pathogenesis the chronic inflammation is involved. Fucoidans are complex polysaccharides from brown seaweeds and some marine invertebrates, composed mainly of L-fucose and sulfate ester groups and minor amounts of neutral monosaccharides and uronic acids. Algae-derived fucoidans are studied intensively during the last years regarding their multiple biological activities and possible therapeutic potential. However, the source, species, molecular weight, composition, and structure of the polysaccharides, as well as the route of administration of fucoidans, could be crucial for their effects. Fucoidan is reported to act on different stages of the inflammatory process: (i) blocking of lymphocyte adhesion and invasion, (ii) inhibition of multiple enzymes, and (iii) induction of apoptosis. In this review, we focused on the immunemodulating and anti-inflammatory effects of fucoidans derived from macroalgae and the models used for their evaluation. Additional insights on the molecular structure of the compound are included.

Etifoxine does not impair muscle tone and motor function in rats as assessed by in vivo and in vitro methods.

The purpose of our study is to evaluate the effects of the translocator protein (TSPO) ligand etifoxine on muscle tone and locomotor activity. In addition, the mechanism of action of etifoxine on the presynaptic membrane and neuromuscular junction is investigated. These effects of etifoxine were examined employing the following methods: 1) in vivo experiments using bar holding test and activity cage test, and 2) comparative in vitro studies with nifedipine on indirectly-elicited twitches of striated abdominal muscle preparations. Etifoxine in doses 50 mg/kg and 100 mg/kg i.p. does not produce any significant changes in locomotor activity and muscle tone of intact rats. Nifedipine (10-5 М) induces a significant decrease in the muscle force of striated muscle preparations. Etifoxine (10-8-10-4 М) has no significant effect on indirectly-elicited twitch tension. Results show that the TSPO ligand etifoxine has no myorelaxant effect. The activation of TSPO is not associated with a reduction in muscle tone and motor impairment. Etifoxine does not affect the presynaptic membrane and its influence on L-type Ca2+-channels is insignificant. Etifoxine does not act as a competitive antagonist of acetylcholine and does not impair the impulse transmission in the neuromuscular junction.

Effect of Bulgarian propolis on the oral microflora in adolescents with plaque-induced gingivitis

ABSTRACT We compared the effect of two therapeutic approaches (marketed toothpaste and addition of Bulgarian propolis extract to the toothpaste) on gingival inflammation, plaque formation and oral microbial flora on Bulgarian adolescents with moderate plaque-induced gingivitis. The participants were divided randomly into two groups of 35 students. The first group was instructed to use marketed toothpaste in their routine oral hygiene. The second group was instructed to add 10 drops of Propolin® to the toothpaste before every brushing. The Gingival index and Plaque index were registered and dental plaque samples were collected on the first visit and on the 20th day of the study. After the treatment, the number of students with Gingival index = 1.1-2.0 in the second group was significantly lower than the respective number in the first group. Neisseria spp. and Streptococcus spp. were present in all samples before and after treatment. The addition of propolis resulted in the complete eradication of Streptococcus mutans, Candida albicans, Fusobacterium varium, Gram-negative cocci, Gram-positive rods, Porphyromonas asaccharolyticus, Prevotella bivia, Prevotella intermedia, Prevotella melani and Streptococcus intermedius. The analyses of Propolin® composition revealed it was a black poplar type propolis and is rich in compounds with pronounced antimicrobial activity. In conclusion, the addition of Bulgarian propolis to the toothpaste improved the gingival health in adolescents with moderate plaque-induced gingivitis and resulted in increased activity against potential periodontal and cariogenic pathogens.

Oral Microbial Flora in Bulgarian Adolescents with Moderate Plaque-induced Gingivitis.

INTRODUCTION: In children and adolescents, the most common periodontal disease is the plaque-induced gingivitis. AIM: The aim of this study was to reveal the bacterial species associated with supragingival plaque of Bulgarian adolescents diagnosed with plaque-induced gingivitis. MATERIALS AND METHODS: Supragingival plaque samples from 70 healthy subjects with moderate plaque-induced gingivitis (37 females and 33 males), aged 12-18 years, were obtained and examined microbiologically. RESULTS: A total of 224 microorganisms were isolated. Gram-negative bacteria were predominant compared to Gram-positive [132 (59%) vs. 92 (41%). CONCLUSION: The most frequently isolated microbiota in our study is part of the normal oral bacterial flora. The presence of anaerobes such as.

Taste Masking of Enalapril Maleate by the Precipitation Method.

BACKGROUND: Taste masking of bitter or unpleasant drugs is an important prerequisite to improve patient compliance, especially for children and elderly patients. We aimed at obtaining taste-masked microparticles intended for incorporation into orodispersible tablets (ODTs). We selected the precipitation method using enalapril maleate (ENA) as a model bitter-tasting drug and Eudragit EPO® as a pH sensitive polymer. AIM: The aim of this study was to obtain microparticles with enalapril maleate by the precipitation method in order to mask the bitter taste of the drug. MATERIALS AND METHODS: Nine models of enalapril maleate ­ Eudragit EPO® microparticles were prepared by the precipitation method at varied drug-polymer ratios. The models were characterized in terms of size, shape, production yield, drug content, encapsulation efficiency and moisture content. Fourier-transformed infrared spectroscopy, powder X-ray diffraction and differential scanning calorimetry were used to analyze possible interactions in the complex. In vitro drug release in simulated salivary fluid and in vivo taste evaluation in rats were realized to prove taste masking. RESULTS: The particle size distribution varied from 266.9 µm to 410.9 µm. The shape of the resulting particles was irregular. The production yield varied from 23.6% to 78.2%. The drug content ranged between 2.3% to 4.8%, encapsulation efficiency increased from 1.6% to 9.0%. In vitro drug release data indicated significant taste masking. CONCLUSION: Some of the obtained microparticles by the precipitation method showed satisfactory taste masking efficiency, which proved the taste masking feasibility of this method.

In vivo investigation of antihyperalgesic and antinociceptive effects of peat formulations.

The aim of this study is to evaluate the antihyperalgesic and antinociceptive effects of two formulations containing peat water extracts using a model of carrageenan-induced hyperalgesia, combined with a test with a mechanical stimulus, and a hot plate test. Rats were divided into seven groups (n = 6) and received local treatment with two peat formulations and two diclofenac formulations dissolved in carbopol gel and Wolff® basis creme, respectively. Carbopol gel, Wolff® basis creme and 0.9 % NaCl without tested substances were used as controls. Both peat formulations exerted an unambiguous antihyperalgesic effect 60 minutes after the treatment. In the hot plate test, the rats treated with the Wolff® basis creme peat formulation showed a tendency to prolonged latency on the first hour. The results could be explained by partial activation of peripheral α2-adrenoceptors and the possible COX-2 suppressive activity.

Ketoprofen-loaded polymer carriers in bigel formulation: an approach to enhancing drug photostability in topical application forms.

The purpose of the study was to investigate the stability and biopharmaceutical characteristics of ketoprofen, loaded in polymeric carriers, which were included into a bigel in a semisolid dosage form. The polymer carriers with in situ-included ketoprofen were obtained by emulsifier-free emulsion polymerization of the monomers in aqueous medium or a solution of the polymers used. The morphological characteristics of the carriers, the in vitro release and the photochemical stability of ketoprofen were evaluated. The model with optimal characteristics was included in a bigel formulation. The bigel was characterized in terms of pH, rheological behavior, spreadability, and in vitro drug release. Acute skin toxicity, antinociceptive activity, anti-inflammatory activity, and antihyperalgesic effects of the prepared bigel with ketoprofen-loaded polymer carrier were evaluated. The carriers of ketoprofen were characterized by a high yield and drug loading. The particle size distribution varied widely according to the polymer used, and a sustained release was provided for up to 6 hours. The polymer mixture poly(vinyl acetate) and hydroxypropyl cellulose as a drug carrier, alone or included in the bigel composition, improved the photostability of the drug compared with unprotected ketoprofen. The bigel with ketoprofen-loaded particles provided sustained release of the drug and had optimal rheological parameters. In vivo experiments on the bigel showed no skin inflammation or irritation. Four hours after its application, a well-defined analgesic, anti-inflammatory, and antihyperalgesic effect was registered. The polymer mixture of poly(vinyl acetate) and hydroxypropyl cellulose as a carrier of ketoprofen and the bigel in which it was included provided an enhanced photostability and sustained drug release.

Retigabine diminishes the effects of acetylcholine, adrenaline and adrenergic agonists on the spontaneous activity of guinea pig smooth muscle strips in vitro.

PURPOSE: The aim of this study is to evaluate the effect of retigabine on the smooth muscle response to acetylcholine, adrenaline, α-and ß-adrenoceptor agonists. METHODS: We studied the change in the spontaneous smooth muscle contraction of guinea pig gastric corpus strips before and after 20-min treatment with 2µM retigabine. We also evaluated the effect of retigabine on the smooth muscle response to 10µM acetylcholine, 1 and 10µM adrenaline, 1µM methoxamine, 0.1µM p-iodoclonidine and 10µM isoproterenol. RESULTS: We observed a significant reduction in the effects of all studied mediators and agonists when they were added to organ baths in the presence of retigabine. Retigabine diminished the effect of acetylcholine on the spontaneous smooth muscle activity. The effect was fully antagonized by XE-991 (Kv7 channel blocker), which supports our hypothesis about the role of KCNQ channels in the registered changes. The increase in the contraction force after adding of 1µM adrenaline, methoxamine, and 0.1µM p-iodoclonidine was also significantly smaller in presence of retigabine. However, comparing the effect of 10µM adrenaline on the contractility before and after treatment with retigabine, we observed increased contractility when retigabine was present in the organ baths. CONCLUSION: A possible explanation for the observed diminished effects of mediators and receptor agonists is that the effect of retigabine on smooth muscle contractility is complex. The membrane hyperpolarization, the interaction between Kv7 channels and adrenoceptors, and the influence on signaling pathways may contribute to the summary smooth muscle response.