Sci. Pharm., Volume 79, Issue 4 (December 2011) – 18 articles , Pages 705-975

The essential oil (EO) of clove bud dried fruits from Eugenia caryophyllus was obtained by a conventional hydrodistillation process in an excellent yield (11.7 %). Its chemical composition was analyzed by GC-MS, identifying eugenol as a main constituent (60.5%). Four eugenol-like molecules, γ-diisoeugenol, hydroxymethyleugenol, dihydroeugenol and 1,3-dioxanylphenol, were synthe-sized using eugenol or isoeugenol as initial precursors under green chemistry protocols. To evaluate the possible antioxidant capacity of eugenol compounds including the clove bud EO, the Trolox® Equivalent Antioxidant Capacity value, obtained by the ABTS^+• radical-cation discoloration method, was employed. The methodology was performed in a UV-Vis reader of 96-well microplates (dilution methodology), using well-known antioxidant agents (BHA, BHT and vitamin E) as reference compounds. It was found that the prepared eugenol derivatives had a more potent free radical scavenger activity than the reference compounds. In particular, the most active molecules, γ-diisoeugenol and 1,3-dioxanylphenol, were ca. 3-fold more potent than vitamin E. Full article

A near infrared (NIR) method able to directly quantify the active content in pharmaceutical powder blends used for manufacturing meloxicam tablets, without any sample preparation, was developed and fully validated. To develop calibration models for the assay of meloxicam in powder blends for tableting, the NIR reflectance spectra of different meloxicam powder blends prepared according to a calibration protocol was analysed using different pre-processing methods by partial last-square regression (PLS) and principal component regression (PCR). The best calibration model was found when partial last-square regression (PLS) was used as regression algorithm in association with Smoothing-Savitsky as pre-processing spectrum method. The trueness, precision (repeatability and intermediate precision), accuracy, linearity and range of application of the developed NIR method were validated according to the International Conference of Harmonization (ICH) and Medicine European Agency (EMA) guidelines and found to be fit for its intended purpose. Full article

Analytical methods were developed for the identification of major degradation products of Ketoconazole, an antifungal agent. The stressed degradation of Ketoconazole drug substance was performed under acid, base, thermal, photo and oxidative stress conditions. The major degradation was observed under acid, base and oxidative stress conditions. The degradation study was performed on Inertsil ODS-3V, length 100 X diameter 4.6 mm, particle size 3 μm column using gradient method. These degradants were identified by LC-MS technique. Full article

A major development over the past two decades has been the realization that free radical induced lipid peroxidation and DNA damage are associated with major health problems, e.g. cancer and ageing. Plant-derived antioxidants are increasingly found beneficial in protecting against these diseases. Celtis australis L. and Celtis occidentalis L. are two plants that have a variety of uses in folk medicine but have not been evaluated before for their antioxidant and cytotoxic properties. Therefore, the extracts of both plants' leaves were investigated for these activities, as well as isolation of the bioactive compounds responsible for the activities. Molecular structures of the compounds were elucidated by UV, HRESIMS, 1D (¹H and ¹³C) and 2D (¹H-¹³C HSQC and ¹H-¹³C HMBC) NMR analyses. The ethanolic and aqueous extracts, n-butanol fractions and the isolated major compound were tested for their antioxidant activity using DPPH radical scavenging assay, xanthine oxidase-induced generation of superoxide radical and lipid peroxidation assay by thiobarbituric acid-reactive substances (TBARS) method using rat tissue homogenates. Cytotoxic activities were studied using standard MTT assay. A novel flavonoid C-triglycoside, 4'''-α-rhamnopyranosyl-2''-O-β-ᴅ-galactopyranosylvitexin, was isolated from both plants' leaves, together with seven known flavonoids. The n-butanol fractions and the major compound 2''-O-β-galactopyranosylvitexin showed significant antioxidant activities, more pronounced than the tested standards BHT and ᴅʟ-α-tocopherol in most tests. All extracts showed variable cytotoxic activities. This study provides strong evidence for the antioxidant and cytotoxic activities of the extracts of Celtis australis L. and Celtis occidentalis L. leaves, which were attributed to the polar n-butanol fractions and the major isolated flavonoid 2''-galactosylvitexin. Full article

Calibration of complex analytical systems is always a difficult task. Nevertheless, a suitable approach has to be designed before the systems can be introduced into routine analysis. In literature, many methods have been described for the purpose of calibrating such systems, but only a few of them deal with capillary elctrophoresis. Here, we want to demonstrate a general approach to how the calibration of this type of analytical instrument becomes feasible. Full article

The antidiabetic activity of both leaves and MJ-treated cell cultures of Morus nigra was evaluated after their oral administration to streptozotocin-induced diabetic rats. The antidiabetic activity of extracts from leaves given to streptozotocin (STZ)-diabetic rats for 10 days increased with increasing doses of leaves extract up to 500 mg/kg/day. The administration of 500 mg/kg/day of leaves extract reduced the concentration of glucose from 370 ± 7.31 mg/dl (control) to 154 ± 6.27 mg/dl, and a significant increase in the insulin level from 11.3 ± 0.31 μU/ml (control) to 14.6 ± 0.43 μU/ml was recorded. Cell suspension cultures were established from the young leaves of Morus nigra cultivated on modified MS medium supplemented with 2.0 mg/l 1-naphthaleneacetic acid (NAA), 0.2 mg/l 6-(furfurylamino)purine (kinetin). The changes in cell weight and flavonoid content were monitored between day zero and 12. The linear increase in fresh weight was found to be parallel to flavonoids production. Cell cultures treated with 100 μM methyl jasmonate for 24 hours showed a noticeable increase in level of flavonoids and significant and more effective hypoglycemic activity than that for extract from leaves. The major flavonoids were isolated by TLC and HPLC and identified as rutin, quercetin, Morusin and cyclomorusin by co-chromatography and mass spectrometry in comparison to samples of authentic reference compounds. Full article

Three new quinazolinobenzodiazepine derivatives, novobenzomalvins A (1), B (2), and C (3), have been isolated as fibronectin expression regulators from Aspergillus novofumigatus CBS117520. The structures of 1 to 3 were established by spectroscopic and physicochemical analysis, and chemical investigation including the total synthesis of 1. Treatment with novo-benzomalvins A (1), B (2), C (3), and N-methylnovobenzomalvin A (5) increased the expression of fibronectin in normal human neonatal dermal fibroblast cells. Full article

The synthesis and evaluation of the anticancer activity of 3’-aryl-5’-arylidene-spiro[3H-indole-3,2’-thiazolidine]-2,4’(1H)-diones and spiro[3H-indole-3,2’-thi-azolidine]-2,4’(1H)-dione-3’-alkanoic acid esters were described. The structure of the compounds was determined by ¹H and ¹³C NMR and their in vitro anticancer activity was tested in the National Cancer Institute. Among the tested compounds, (5'Z)-5'-(benzylidene)-3'-(4-chlorophenyl)spiro[3H-indole-3,2'-thia-zolidine]-2,4'(1H)-dione (IIa) and (5'Z)-3'-(4-chlorophenyl)-5'-[4-(1-methylethyl)-benzylidene]spiro[3H-indole-3,2'-thiazolidine]-2,4'(1H)-dione (IIb) were superior to other related compounds. Full article

A sensitive and simple HPLC/UV method has been developed and validated for the determination of two potential genotoxic impurities, namely methyl p-toluenesulfonate (MPTS) and ethyl p-toluenesulfonate (EPTS) at trace levels in Pemetrexed sodium API. Applying the concept of threshold of toxicological concern (TTC), a limit of 3 ppm each for both genotoxins was calculated based on the maximum daily dose of API. A reversed phase LC method using UV detection was developed and validated. The method was found to be specific and selective for the application. The limit of detection (LOD) and limit of quantitation (LOQ) for both MPTS and EPTS was found to be 0.15 ppm (0.009 μg mL⁻¹) and 0.5 ppm (0.03 μg mL⁻¹), respectively, with respect to sample concentration. The calibration curves of MPTS and EPTS were linear over the concentration range from LOQ to 6 μg/mL. The method was found to be specific, precise, linear and accurate and has been successfully applied to determine the two genotoxins in commercial batches of the API. Full article

A series of dibenzo[b,e]ox(thi)epin-11(6H)-one O-benzoyloximes has been synthesized and structurally elucidated by means of IR, ¹H-NMR, ¹³C-NMR, MS, and elemental analysis. The newly developed compounds were screened at concentrations of 200–25 μg/mL for their antibacterial activity against Gram+ve organisms such as Methicillin-Resistant Staphylococcus Aureus (MRSA), Gram–ve organisms such as Escherichia coli (E. coli), and at the same concentration range for their antifungal activity against fungal strain Aspergillus niger (A. niger) by the cup plate method. Ofloxacin and ketoconazole (10 μg/mL) were used as reference standards for antibacterial and antifungal activity, respectively. The dibenzo[b,e]oxepines 6a–c and 6e–h showed low antimicrobial activity (MIC 125–200 μg/mL) compared to the reference substances, whereas a major improvement (MIC 50–75 μg/mL) was achieved with the synthesis of the corresponding bromomethyl derivative 6d. Moreover, replacement of oxygen by its bioisosteric sulfur led to isomeric dibenzo[b,e]thi-epine derivatives 6g,h which significantly exhibited higher antimicrobial activity (MIC 25–50 μg/mL) against all tested culture strains used in the present study, demonstrating that a change of chemical class from dibenzo[b,e]oxepine to dibenzo[b,e]thiepine significantly improves the antimicrobial activity. Further variation, such as the oxidation of the thiepine sulfur to the corresponding isomeric dibenzo[b,e]thiepine 5,5-dioxide derivative 9, comparatively failed to exhibit high activity (MIC 200 μg/mL) against S. aureus, E. coli or A. niger. Full article

Lopinavir is a new specific and potent HIV-1 protease inhibitor. A simple and rapid Reverse Phase High-Performance Liquid Chromatographic method using UV detection was developed and validated for the analysis of lopinavir in rat plasma under isocratic conditions. The method involves a single step protein precipitation technique. The detector response was linear over the concentration range of 250 to 4000 ng mL⁻¹. High recovery ranging from 97.5 to 101.2 percent was obtained which precludes the use of internal standard. The developed method was validated as per standard guidelines. Validation of the developed method demonstrated accuracy, precision and selectivity of the proposed method. The drug was found to be stable under various processing and storage conditions. This rapid and cost-effective method was successfully applied in the estimation of lopinavir and determination of various pharmacokinetic parameters during post intravenous bolus administration of the drug in rats. The developed method can be suitably employed in preclinical pharmacokinetic evaluation of new formulations designed to improve the bioavailability of lopinavir. Full article

Many anti-tumor drugs function by intercalating into DNA. The xanthine alkaloid caffeine can also intercalate into DNA as well as form π-π molecular complexes with other planar alkaloids and anti-tumor drugs. The presence of caffeine could interfere with the intercalating anti-tumor drug by forming π-π molecular complexes with the drug, thereby blocking the planar aromatic drugs from intercalating into the DNA and ultimately lowering the toxicity of the drug to the cancer cells. The cytotoxic activities of several known DNA intercalators (berberine, camptothecin, chelerythrine, doxorubicin, ellipticine, and sanguinar-ine) on MCF-7 breast cancer cells, both with and without caffeine present (200 μg/mL) were determined. Significant attenuation of the cytotoxicities by caffeine was found. Computational molecular modeling studies involving the intercalating anti-tumor drugs with caffeine were also carried out using density functional theory (DFT) and the recently developed M06 functional. Relatively strong π–π interaction energies between caffeine and the intercalators were found, suggesting an “interceptor” role of caffeine protecting the DNA from intercalation. Full article

An isocratic ion pair RP-HPLC method with UV-Vis detection has been developed and validated for simultaneous analysis of 4-nitrophenol (PNP), 4-nitrophenyl β-glucuronide (PNP-G), and 4-nitrophenyl sulfate (PNP-S) in rat bile samples using 4-ethylphenol (ETP) as internal standard. Chromatographic separation was achieved on a C18 column by isocratic elution with a mobile phase consisted of methanol-0.01 M citrate buffer pH 6.2 (47:53 v/v) containing 0.03 M TBAB. The flow rate was 1.0 ml min⁻¹, the detection was affected at 290 nm. Calibration plots were generated over the concentration range 1–100 μM PNP, PNP-G, PNP-S with a common lower limit of quantification of 2.5 μM. Intra- and inter-day precision and repeatability were determined at six different concentrations. Results obtained by application of the method for determination of PNP, PNP-G and PNP-S in bile fractions collected during intestinal perfusion of PNP in hyperglycemic rats are presented. Full article

The relative lack of clinical success with conventional anticancer agents may be due in part to the traditional concept of cancer being a biological state rather than a dynamic process. Redefining cancer as a dynamic disease commencing with carcinogenesis introduces the possibility of chemoprevention. Retinoids offer the promise of a therapeutic option based on differentiation of premalignant as well as malignant cells. Research to date has concentrated on the use of exogenous retinoids in cancer. Although this research continues with new retinoid derivatives, an alternative approach to overcoming the drawbacks associated with exogenous retinoids has been to increase the levels of endogenous retinoic acid (RA) by inhibiting the cytochrome P450- mediated catabolism of RA using a novel class of agents known as retinoic acid metabolism blocking agents (RAMBAs which increase the level of endogenous retinoic acid (RA) within the tumor cells by blocking their metabolism. This approach presents several theoretic advantages. In the present study a wide range of established P-450 inhibitors has been screened to examine their inhibitory activity on all-trans-Retinoic acid (ATRA) metabolism. Forty-one known P450 inhibitors were tested for their inhibitory activity against RA metabolism. Most of them are nitrogen-containing compounds. The results showed that among these compounds only six compounds (N-benzyl-2-phenylethanamine, itraconazole, chlorpromazine, 5-chloro-1,3-benzoxazol-2-amine, proadifen and furazolidone) showed inhibition of RA metabolism which was > 50%. Ketoconazole and liarozole were also screened as standard potent inhibitors in the same system and gave 87.5% and 89% inhibition, respectively. The results indicate that mostly azoles with substituents in positions other than the 1-position on the ring are very weak inhibitors of RA metabolism. The most effective inhibitors (ketoconazole, itraconazole, bifonazole and clotrimazole) are 1-substituted and possess relatively large aromatic groups in the molecule. 1-Substituted imidazoles bind to cytochrome P-450 with a very high affinity but substitution in the other position of the imidazole decreases the binding affinity. Full article

A stability-indicating reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed for the determination of sodium bisulfate (SB), an antioxidant, in injectable dosage form. The chromatographic separation was achieved on a Zorbax CN (250 mm × 4.6 mm, 5 μm) column, with a mobile phase consisting of a buffer mixture of 0.03 M tetrabutylammonium hydrogen sulfate, 0.01 M potassium dihydrogen orthophosphate, and acetonitrile at a ratio of 70:30 (v/v) and a flow rate of 0.7 mL/min. The eluted compound was monitored at a wavelength of 215 nm using a UV detector. The method described herein separated sodium bisulfite from all other formulation components within a run time of 10 min. The method also generated linear results over an SB concentration range of 10 to 990 μg/mL, and the limit of quantification was found to be 10 μg/mL. The stability indicating capability of the method was established by performing forced degradation experiments. The RP-HPLC method that was developed was validated according to the International Conference on Harmonization (ICH) guidelines. This method was successfully applied in the quantitative determination of SB in a stability study of Amikacin sulfate injection. The procedure described herein is simple, selective, and reliable for routine quality control analysis as well as stability testing. Full article

A simple stability indicating reversed-phase HPLC method was developed and subsequently validated for estimation of Cefpirome sulphate (CPS) present in pharmaceutical dosage forms. The proposed RP-HPLC method utilizes a LiChroCART-Lichrosphere100, C18 RP column (250 mm × 4mm × 5 μm) in an isocratic separation mode with mobile phase consisting of methanol and water in the proportion of 50:50 % (v/v), at a flow rate 1ml/min, and the effluent was monitored at 270 nm. The retention time of CPS was 2.733 min and its formulation was exposed to acidic, alkaline, photolytic, thermal and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. The described method was linear over a range of 0.5–200μg/ml. The percentage recovery was 99.46. F-test and t-test at 95% confidence level were used to check the intermediate precision data obtained under different experimental setups; the calculated value was found to be less than the critical value. Full article

The prostanoid receptor EP1 is a G-protein-coupled receptor (GPCR) known to be involved in a variety of pathological disorders such as pain, fever and inflammation. These receptors are important drug targets, but design of subtype specific agonists and antagonists has been partially hampered by the absence of three-dimensional structures for these receptors. To understand the molecular interactions of the PGE2, an endogen ligand, with the EP1 receptor, a homology model of the human EP1 receptor (hEP1R) with all connecting loops was constructed from the 2.6 Å resolution crystal structure (PDB code: 1L9H) of bovine rhodopsin. The initial model generated by MODELLER was subjected to molecular dynamics simulation to assess quality of the model. Also, a step by step ligand-supported model refinement was performed, including initial docking of PGE2 and iloprost in the putative binding site, followed by several rounds of energy minimizations and molecular dynamics simulations. Docking studies were performed for PGE2 and some other related compounds in the active site of the final hEP1 receptor model. The docking enabled us to identify key molecular interactions supported by the mutagenesis data. Also, the correlation of r²=0.81 was observed between the Ki values and the docking scores of 15 prostanoid compounds. The results obtained in this study may provide new insights toward understanding the active site Sci Pharm. 2011; 79: 793–816 conformation of the hEP1 receptor and can be used for the structure-based design of novel specific ligands. Full article

Lipid-based formulations encompass a diverse group of formulations with very different physical appearance, ranging from simple triglyceride vehicles to more sophisticated formulations such as self-emulsifying drug delivery systems (SEDDS). Lipid-based drug delivery systems may contain a broad range of oils, surfactants, and co-solvents. They represent one of the most popular approaches to overcome the absorption barriers and to improve the bioavailability of poorly water-soluble drugs. Diversity and versatility of pharmaceutical grade lipid excipients and drug formulations as well as their compatibility with liquid, semi-solid and solid dosage forms make lipid systems most complex. Digestion of triglyceride lipids, physicochemical characteristics and solubilisation of lipid digestion products as well as intestinal permeability are some of the variable parameters of such formulations. Furthermore, among the factors affecting the bioavailability of the drug from lipid-based formulations are the digestion of lipid, the mean emulsion droplet diameter, the lipophilicity of the drug and the type of lipids. The solubility of the Active Pharmaceutical Ingredient in the Lipid System, the desorption /sorption isotherm and the digestibility of lipid vehicle are important issues to be considered for formulations of isotropic lipid formulations. This review also describes the fate of lipid formulations in the gut and the factors influencing the bioavailability from lipid-based formulations. Novel formulation systems and currently marketed products conclude this review. Full article