Sci. Pharm., Volume 87, Issue 2 (June 2019) – 7 articles

The aim of this work was to assess the analgesic effect of three Vitis vinifera L. leaf extracts and the anti-inflammatory effect of three gels obtained from Aesculus hippocastanum L. seed extracts using animal models, as a preliminary study for the future development of topical preparations based on the combination of extracts with synergistic therapeutic effects on hemorrhoid disease. The analgesic effect was determined by means of the writhing test in mice. The anti-inflammatory effect was determined after administration of carrageenan or kaolin in the rat paw. Extraction using glycerol yielded the highest amounts of flavonoids for both V. vinifera leaves (37.27 ± 1.174 mg/L) and A. hippocastanum seeds (53.48 ± 0.212 mg/L). The highest total phenolic contents were registered for the V. vinifera 20% ethanolic extract (615.3 ± 34.44 mg/L) and for the A. hippocastanum glycerolic extract (247.8 ± 6.991 mg/L). The writhing test revealed that the V. vinifera ethanolic extract induced the most efficient analgesia (57.20%, p < 0.01), better than that induced by the positive control. In the carrageenan inflammation model, only the gel obtained from the A. hippocastanum glycerolic extract significantly reduced paw edema (17.27%, p < 0.05). An anti-inflammatory effect was also observed in the kaolin inflammation model but was not statistically significant (10.12%, p > 0.05). Our findings indicate that V. vinifera and A. hippocastanum extracts may have potential uses for the treatment of pain and inflammation associated with hemorrhoid disease. Full article

Diabetes is a major metabolic disorder whose prevalence is increasing daily. Medicinal plants have played an important role in the prevention and treatment of type 2 diabetes via prophylactic and therapeutic management. In this study, Mangifera Indica leaf (MIL) extract was investigated for its promising anti-diabetic activity via an in vitro model. It was found that MIL extract possessed significant inhibition on alpha-amylase activity up to (51.4 ± 2.7)% at a concentration of 200 µg/mL. Moreover, glucose adsorption capacity of MIL was identified at (2.7 ± 0.19) mM glucose/g extract. Furthermore, the extract caused a significant increase in glucose uptake up to (143 ± 9.3)% in LO-2 liver cells. Notably, MIL extract was effective in scavenging (63.3 ± 2.1)% 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and (71.6 ± 4.3)% 2,2-azinobis-3-ethyl benzothiazoline-6-sulfonic acid (ABTS)⁺ radicals and inhibiting (66 ± 4.9)% NO production from RAW264.7 cells without any cytotoxicity effects. Accordingly, M. indica leaves are suggested as a promising material for development of hypoglycemic products. Full article

According to our quantum and chemical calculations 4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxylic acid imidazolide is theoretically almost as reactive as its 2-carbonyl analog, and it forms the corresponding N-pyridyl-4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxamides with many aminopyridines. However, in practice, the sulfo group introduces significant changes at times and prevents the acylation of sterically hindered amines. One of these products was 2-amino-6-methylpyridine. Thus, it has been concluded that aminopyridines interact with imidazolide in aromatic form where the target for the initial electrophilic attack is the ring nitrogen. To confirm the structure of all substances synthesized, ¹H-NMR spectroscopy and X-ray diffraction analysis were used. From X-ray diffraction data it follows that in the crystalline phase the carbonyl and sulfo group may occupy different positions with respect to the plane of the benzothiazine bicycle: this position may be unilateral, typical for 4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxamides, versatile, and not yet encountered in compounds of this type. A comparison of these data with the results of the pharmacological screening conducted on the standard model of carrageenan inflammation showed that the N-pyridylamides of the first group demonstrated a direct dependence of their analgesic and anti-inflammatory activity on the mutual arrangement of the planes of the benzothiazine and pyridine fragments. The new molecular conformation of the benzothiazine nucleus provides a sufficiently high level of analgesic (but not anti-inflammatory) properties in all N-pyridylamides of the second group with an extremely weak dependence on the spatial arrangement of the pyridine cycle. All substances presented this article proved themselves in varying degrees as analgesics and antiphlogistics. Moreover, two of them—N-(5-methylpyridin-2-yl)- and N-(pyridin-3-yl)-4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxamides—exceeded the most effective drug of oxicam type Lornoxicam by these indicators. Full article

In the last decade, high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with electrospray ionization (ESI) has been widely used for determining low concentrations of steroids, and derivatization has often been employed to enhance detection. In the present study, endogenous steroids were extracted using a Strata-XL polymeric reverse phase cartridge. The isolated steroids were reacted with 2-hydrazino-1-methylpyridine (HMP) at 50 °C for 30 min. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used in a positive mode with multiple reaction monitoring (MRM) for the quantification of testosterone (T) and its precursor, dehydroepiandrosterone (DHEA), in saliva samples collected from twenty young Saudi professional soccer players. The analytes were separated on an ACE Ultracore 2.5 Superphenylhexyl column (150 × 3.0 mm id). The extraction recovery during the pre-treatment was >89% and gave <±20% for inter- and intra-assay precision and accuracy. The limits of quantification (LOQ) were found to be 20 pg/mL for (T and DHEA) and 50 pg/mL for Epitestosterone (EPI). The results showed no significant variation in the concentration of T between pre and post training, whereas DHEA was significantly increased after short-term exercise. These results could indicate that there is no correlation between T and its precursor DHEA level following short term physical activity. EPI concentrations could not be detected with a LOQ of 50 pg/mL in the saliva samples. Full article

In order to detect new structural and biological patterns in a series of hetaryl-3-carboxylic acid derivatives, the optically pure (S)- and (R)-enantiomers of N-(1-arylethyl)-4-methyl- 2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxamides, their true racemates, and mechanical racemic mixtures have been synthesized in independent ways. The particular features of the ¹Н- and ¹³С-NMR spectra of all synthesized substances, liquid chromato-mass spectrometric behavior thereof under electrospray ionization conditions, and also the results of polarimetric and X-ray diffraction studies have been discussed. Pharmacological screening on a model of carrageenan inflammation has found a clear relationship between the spatial structure of the studied objects and biological activity thereof. Enantiomers with chiral centers having (S)-configuration showed weak inhibition of pain and inflammatory reactions, while their mirror (R)-isomers exhibited very powerful analgesic and antiphlogistic properties under the same conditions, with the level of specific activity exceeding that of Lornoxicam and Diclofenac. Taking obtained data into account, a noticeable decrease in the activity of mechanical racemic mixtures, consisting of one-half of the “wrong” (S)-enantiomers, is quite natural. The true racemate of N-(1-phenylethyl)-amide proved itself in a similar way, while 4-methoxy-substituted analog thereof stood out against this background with unexpectedly high analgesic and anti-inflammatory activities. A comparative analysis of X-ray diffraction data has found that crystalline and molecular structure of racemic N-[1-(4-methoxyphenyl)ethyl]-4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxamide is completely different from that of the original enantiomers and, moreover, very unusual for racemates. Obviously, it is the factor determining the unique character of the biological effects of the said substance. Full article

The aim of this study was to investigate the acute and subchronic toxicity of a film formulation that combines κ-Carrageenan and konjac glucomannan for soft capsule application. For the acute toxicity study, a dose of 2000 mg/kg body weight (bw) of the film suspension was administered orally to rats. The animals were observed for toxic symptoms and mortality daily for 14 days. In a subchronic toxicity study, the film suspension, at doses of 10, 30 and 75 mg/kg bw for 28 days, were orally administered to rats. After 28 days, the rats were sacrificed for hematological, biochemical and histological examination. In the acute toxicity study, neither signs of toxicity nor death among the rats were observed for up to 14 days of the experimental period. The results of the subchronic toxicity study show that there were no significant changes observed in the hematology and organ histology. Some alterations to the relative organ weight and blood biochemistry were observed, but they were considered to be temporary effects and not an indication of toxic effects. The overall findings of this study indicate that the film formulation of κ-Carrageenan and konjac glucomannan is non-toxic up to a dose of 75 mg/kg bw, which could be considered a safe dose for soft capsule application. Full article

Rhodiola rosea has been used in folk medicine as ethanolic macerates for a long time. This study aims to provide a quantitative and qualitative analysis and comparison of different ethanolic Rhodiola rosea rhizome macerates (35%, 70%, and 96% v/v) and accelerated solvent extraction (ASE) extracts prepared with 85% methanol, in order to shed light on the effectivity of different extraction methods. Extract samples were analyzed by UHPLC-DAD-ESI-MSⁿ on a ZORBAX SB-C18 column (100 × 2.1 mm, 1.8 μm) with a mobile phase consisting of water + 0.1% formic acid and acetonitrile. Qualitative analysis lead to the tentative identification of 18 compounds: Two cyanogenic glycosides (rhodiocyanoside A, lotaustralin), three phenylethanoids (salidroside, viridoside, 2-phenylethyl-vicianoside), two procyanidin and catechin derivatives (epigallocatechin-epigallocatechin gallate, epigallocatechin-3-O-gallate), five phenylpropanoids (cinnamyl alcohol, rosarin, rosavin, rosin, cinnamyl-(6’-O-β-d-xylopyranosyl)-O-β-glucopyranoside), two monoterpene alcohols (rhodioloside E, rosiridin) and four flavonols (rhodionidin, rhodiosin, rhodionin, kaempferol). Quantity was determined for salidroside, cinnamyl alcohol and its three major glycosides (rosarin, rosavin, rosin), as well as three flavonols (rhodionidin, rhodiosin, rhodionin). Methanolic ASE proved to be the superior extraction method for different substance groups. For macerates, high ethanol content increased yield and lowered hydrolysis of glycosides during extraction, but ethanolic macerates still showed low reproducibility and high fluctuations in quantity of marker compounds salidroside and rosavins, as well as flavonols. Rhodiola rosea rhizomes of wild origins seemed to underly great variability in chemical composition dependent on grow site. Full article