Open Access Original Research Article

Changes in the Oxidative Stress Biomarkers in Liver of Streptozotocin-diabetic Rats Treated with Combretum lanceolatum Flowers Extract

Carlos Roberto Porto Dechandt, Damiana Luiza Pereira De Souza, Juliany Torres Siqueira, Mayara Peron Pereira, Renata Pires De Assis, Virginia Claudia Da Silva, Paulo Teixeira De Sousa Junior, Iguatemy Lourenço Brunetti, Claudia Marlise Balbinotti Andrade, Nair Honda Kawashita, Amanda Martins Baviera

Journal of Pharmaceutical Research International, Page 2340-2356
DOI: 10.9734/BJPR/2014/13558

Aims: The study investigated the in vivo antioxidant activity and the in vitro radical scavenging capacity of the Combretum lanceolatum Pohl (Combretaceae) flowers ethanolic extract (ClEtOH) in streptozotocin-diabetic rats.
Place and Duration of Study: Department of Chemistry, Federal University of Mato Grosso, Cuiabá, Brazil; between February 2012 and December 2012.
Methodology: Male Wistar rats were divided into four groups: Normal rats treated with water/vehicle (N); diabetic rats treated with water (DC); diabetic rats treated with 250 mg/kg (DT250) or with 500mg/kg (DT500) of ClEtOH. After 21 days of treatment, liver samples were used for the analysis of the oxidative stress biomarkers and activity of antioxidant enzymes. In vitro radical scavenger capacity was investigated by the following methods: DPPH radical scavenging, ABTS radical cation decolorization and crocin bleaching assays.
Results: Significant oxidative stress was observed in liver of DC, since the malondialdehyde (MDA, biomarker of lipoperoxidation) levels were increased in comparison with N. Increased activities of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were also observed in DC, which could represent a compensatory mechanism against oxidative stress. Glutathione (GSH) levels were lower and similar between N and DC. The MDA levels were significantly decreased in liver of rats from DT250 and DT500, reaching levels similar those of N, suggesting that ClEtOH prevented lipoperoxidation. The treatment of diabetic rats with ClEtOH also increased the GSH levels, as well as increased the GSH-Px activity, and did not change the SOD activity. The results of in vitro radical scavenging capacity indicated that ClEtOH is highly active.
Conclusion: These findings indicate that ClEtOH has antioxidant properties in liver of diabetic rats, decreasing lipoperoxidation and increasing the endogenous antioxidant responses. Both the antihyperglycemic effect and the capacity to scavenge free radicals may be related to the antioxidant activity of ClEtOH in diabetes.

Open Access Original Research Article

Antibacterial and Anticandidal Activities of New Flavonoids from Streptomyces sp. HK17; an Endophyte in Curcuma longa Linn

Thongchai Taechowisan, Srisakul Chanaphat, Wanwikar Ruensamran, Waya S. Phutdhawong

Journal of Pharmaceutical Research International, Page 2357-2369
DOI: 10.9734/BJPR/2014/13326

Aims: The purpose of this study was to investigate the antibacterial and anticandidal activities of new flavonoids from Streptomyces sp. HK17 which was isolated from the root tissue of Curcuma longa Linn.
Study Design: Experimental study.
Place and Duration of Study: The study was carried out at the Department of Microbiology and Department of Chemistry, Faculty of Science, Silpakorn University, between February and May 2014.
Methodology: The major active ingredients from the crude extract were purified by silica gel column chromatography, thin-layer chromatography. The diameters of the zones of inhibition and the Minimum Inhibitory Concentration (MIC) were determined using the paper disc diffusion and the microbroth dilution methods respectively.
Results: The crude extract and purified compounds were tested for their antibacterial activity against Staphylococcus aureus ATCC25932, Bacillus cereus ATCC7064 and Bacillus subtilis ATCC6633, Escherichia coli ATCC10536 and Pseudomonas aeruginosa ATCC27853 and anticandidal activity against Candida albicans ATCC190088. The crude extract showed the highest activity against S. aureus and C. albicans, with MIC values of 32 µg/ml. The purified compounds 3 showed the lowest MIC (32 µg/ml) and Minimum Microbicidal Concentration (MMC) (128 µg/ml) against S. aureus and C. albicans with corresponding large diameter of the zone of inhibitions (25.5 and 25.2 mm respectively).
Conclusion: This study has shown that the new flavonoids were first isolated and identified. These flavonoids produced by Streptomyces sp. HK17 have potential in antibacterial and anticandidal activities.

Open Access Original Research Article

RP-HPLC and HPTLC Methods for Simultaneous Estimation of Metformin Hydrochloride and Vildagliptin from Bulk and Marketed Formulation: Development and Validation

A. R. Shirode, P. D. Maduskar, M. S. Deodhar, V. J. Kadam

Journal of Pharmaceutical Research International, Page 2370-2386
DOI: 10.9734/BJPR/2014/12820

The reversed-phase high performance liquid chromatography (RP-HPLC) and high performance thin layer chromatography (HPTLC) methods for simultaneous estimation of Metfomin Hydrochloride (MET) and Vildagliptin (VLD) in bulk and their marketed combined dosage form were developed.
For RP-HPLC, separation was carried out using HiQsil C18HS (4.6mmø×250mm) analytical column and detection was carried out using variable wavelength detector. The mobile phase was composed of phosphate buffer (pH adjusted to 6 using 3M KOH): methanol: acetonitrile in the ratio of 50:30:20 v/v/v. Flow rate was kept at 0.8ml/min. The drugs- MET and VLD were retained at 3.7 minutes and 4.8 minutes respectively.
The HPTLC method was developed using Camag HPTLC system. Silica Gel 60GF254 precoated TLC plates were used as stationary phase. The mobile phase was ammonium acetate in methanol (1% w/v): Toluene; (10:0.5). The detection of spots was carried out densitometrically at 214 nm in absorbance mode. The Rf values for MET and VLD were found to be 0.44 and 0.55 respectively.
Performance characteristics of both of these RP-HPLC and HPTLC methods for simultaneous estimation of MET and VLD in bulk and their marketed combined dosage form were statistically validated as per the recommendations of ICH guidelines of analytical method validation.
The RP-HPLC method was found to be linear across concentration range of 10-60µg/mL for MET and VLD respectively. For RP-HPLC the LOD values for MET and VLD were 1.09µg/ml and 1.70µg/ml respectively and LOQ values for MET and VLD were 3.32µg/ml and 5.15µg/ml respectively.
The HPTLC method was found to be linear with across the range 1000-5000ng/spot and 500-2000ng/spot for MET and VLD respectively. For HPTLC the LOD values for MET and VLD were 17.22ng/spot and 34.60ng/spot respectively and LOQ values for MET and VLD were 52.20ng/spot and 104.85ng/spot respectively.
Both of these RP-HPLC and HPTLC methods were found to be simple, specific, linear, accurate, precise and robust, hence any of these methods can be conveniently adopted for routine analysis of the formulations containing MET and VLD, for their simultaneous estimation.

Open Access Original Research Article

Chitosan Nanoparticles for Topical Co-administration of the Antioxidants Glutathione and Idebenone: Characterization and In vitro Release

Lucia Montenegro, Adriana Trapani, Paola Fini, Delia Mandracchia, Andrea Latrofa, Nicola Cioffi, Laura Chiarantini, Giusi Giada Picceri, Serena Brundu, Giovanni Puglisi

Journal of Pharmaceutical Research International, Page 2387-2406
DOI: 10.9734/BJPR/2014/8641

Aims: The aim of this study was to explore the potential of novel nanoparticles (NPs) intended for topical administration of the hydrophilic antioxidant Glutathione and the lipophilic Idebenone. Glutathione was introduced into the NPs using two approaches: i) covalently bonded to Chitosan; ii) physically complexed with Idebenone and Sulfobutylether-ï¢-cyclodextrin.
Methodology: NPs were formulated using the ionic gelation technique, by dissolving the polysaccharide-forming matrix (Chitosan, Glycol chitosan, Glutathionyl Chitosan) in water or in slightly acidic solution. Idebenone was physically entrapped whereas glutathione was either physically entrapped or covalently bonded to chitosan.
Physicochemical characterization of the resulting NPs included size, zeta potential measurements, antioxidant association efficiency, differential scanning calorimetry (DSC) and stability studies. Antioxidants in vitro release from the most stable NPs was assessed with Franz diffusion cells, and the in vitro antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical test. NP cytotoxicity was assessed on immortalized human keratinocytes (HaCaT) cell line.
Results: The NPs showed smaller particle size in acidic solution than in aqueous medium, whereas zeta potential values were always positive, irrespective of the medium. Stability studies led to the choice of the aqueous formulation where Glutathione was covalently bonded to Chitosan for this study. DSC highlighted amorphization of Idebenone in these NPs. In vitro release studies showed that only Idebenone was released from the NPs. The antioxidant activity test revealed a strong effect (close to 100%) of Idebenone loaded into NPs while its aqueous solution showed no activity. No cytotoxicity in human keratinocytes was observed for the investigated NPs.
Conclusion: The results of this study suggest that Idebenone can be loaded into a hydrophilic delivery system without organic solvents, often used for its solubilization, possessing high antioxidant activity. Therefore, these nanocarriers represent a promising strategy for the design of formulations for topical treatments with antioxidants.

Open Access Original Research Article

Prevalence and Antimicrobial Susceptibility Pattern of Urinary Bacterial Pathogens Isolated from Diabetes Mellitus Patients in Uyo, Nigeria

Sunday Olajide Awofisayo, Ayodeji Akeem Agboke, Ekaette Dominic Nkanang

Journal of Pharmaceutical Research International, Page 2407-2416
DOI: 10.9734/BJPR/2014/11205

This study was aimed at determining the effect of hyperglycaemia and glysuria on the antimicrobial susceptibility of pathogens isolated in UTI. 236 patients visiting the out-patient department of University of Uyo Health Centre presenting with symptoms of urinary tract infection were recruited post oral interview. Fasting blood sugar (FBS) levels over three successive days were performed for the enlisted participants. Urine specimen was collected from each participant for determination of glucose in urine, microbiology, culture and sensitivity. Oral interview revealed patients’ confessed past medical history of diabetes (PMHD) and no past medical history of diabetes (NPMHD) of 105 (44.5%) and 131 (55.5%) respectively. Mean fasting blood sugar (MFBS) ≥7.0 was observed in 12.7% participants and glysuria in 81 (34.3%). Microbiological screening revealed bacteriuria in 88.1% and significant infection (>100,000 bacteria/ml) in 46.2%. The bacteria isolate and (frequency of occurrence percent) were Klebsiella spp. 3 (2.6%), Staphylococcus aureus 36 (26.1%), Escherichia coli 79 (66.4%) and Proteus spp. 2(1.7%). E. coli isolated in the DM group had no significant difference in the susceptibility pattern to the cephalosporin or the flouroquinolone antibacterial compared with the control group. S. aureus however revealed significantly higher susceptibility to the cephalosporin antibacterial agents but no difference with the flouroquinolone when compared with the control group (P<0.05). There was strong indication for laboratory antimicrobial sensitivity determinations in DM for the appropriate choice of drug treatment.

Open Access Original Research Article

Effect of Formulation Variables on the Release of Letrozole from Natural Biodegradable Polymeric Implants

S. Mehbuba Hossain, S. Islam, M. Saha, S. Islam

Journal of Pharmaceutical Research International, Page 2417-2435
DOI: 10.9734/BJPR/2014/12432

Aim: Letrozole, a non-steroidal aromatase inhibitor, prevents the body from producing its own estrogen. The objective of the present study was to explore the fabrication and evaluation of natural biodegradable polymeric Letrozole implant for long term drug release targeting postmenopausal women with metastatic breast cancer.
Methodology: The effect of different formulation variables i.e. different types of excipients and different hardening times (6 hrs, 12 hrs and 24 hrs) with exposure to formaldehyde vapour was investigated on drug loading efficiency and drug release profile. The result of in-vitro dissolution study was fitted to different kinetic models to evaluate the kinetic data.
Results: Letrozole release was studied for 10 to 19 days with some excipients. The in vitro Letrozole release from Gelatin-Sodium Alginate biodegradable polymeric implant was maximum, about 19 days, where Cetyl alcohol was incorporated as excipient. The release kinetics was explored and explained using Higuchi, zero and first order while the mechanism of release was confirmed with Korsmeyer-peppas model. Implants were found to follow Higuchi model the best in most cases. Good correlations were also obtained with Korsmeyere-Peppas model. According to these models, the drug released from implants were of diffusion controlled, where the drug was found to leave the matrix through pores and channels formed by entry of dissolution medium.
Conclusion: The addition of different excipients and variation in hardening times were found to influence the drug loading efficiency and drug release significantly. Further investigation would confirm its potential in breast cancer therapy.

Open Access Original Research Article

Preparation, Characterization and Optimization of Poloxamer Solid Dispersions of a Poorly Water Soluble Drug Aprepitant

Rabia Dhillon, Ritu Ojha, Neena Bedi

Journal of Pharmaceutical Research International, Page 2436-2454
DOI: 10.9734/BJPR/2014/10561

Aim: The aim of the present investigation was to enhance the dissolution rate of a poorly water soluble drug, aprepitant, by preparation of solid dispersion with hydrophilic carrier, poloxamer 188, using solvent evaporation method.
Place and Duration of Study: Department of Pharmaceutical Sciences and Department of Emerging Life Sciences, Guru Nanak Dev University, Amritsar, Punjab, between August 2012 and July 2013.
Study Design: Designs of experiments were carried out with two input variables, drug to carrier ratio (X1) and amount of solvent (X2). A total of 7 experiments were performed (4 factorial runs and 3 centre points) as per full factorial design. Percent dissolution efficiency at 60 min (DE60) was selected as the response variable. Pareto chart along with half probability plot were studied for determining the most significant process variables, which were modelled using ANOVA.
Methodology: Solid dispersions of aprepitant with poloxamer 188 were prepared using the solvent evaporation method and studied systematically using an optimization technique. A 22 full factorial design approach was used for the optimization of process variables on dissolution characteristics. The optimized solid dispersion was characterized by dissolution studies, Fourier Transform Infrared Spectroscopy, Differential Scanning Calorimetry, X-ray powder Diffraction studies and Scanning Electron Microscopy.
Results: The results of the experimental study confirm that the factors like drug to carrier ratio and solvent amount significantly influence the dependent variable DE60. A high level of drug to carrier ratio and low level of solvent amount were found to be suitable for maximum DE60. The use of factorial design approach helped in the optimization of the solid dispersion. The characterization of optimized solid dispersion (F5) demonstrated that the decrease in crystallinity of aprepitant and poloxamer 188 might be responsible for the enhanced dissolution rate. Analysis of dissolution data indicated the best fitting with first-order model.
Conclusion: Dissolution enhancement of aprepitant was successfully obtained by preparing its solid dispersion with poloxamer 188 using solid evaporation method.

Open Access Original Research Article

Uterine Relaxation Potential of Ethanol Leaf Extract of Moringa oleifera Lam. VIA the Muscarinic Receptor Pathway

N. S. Ijioma, K. G. Madubuike, O. N. Nwankudu, C. O. Nwosu, C. U. Emelike

Journal of Pharmaceutical Research International, Page 2455-2462
DOI: 10.9734/BJPR/2014/12140

Aim: The effect of Ethanol Leaf Extract of Moringa oleifera (ELMO) on uterine smooth muscles of non- pregnant female rats was studied in vitro with a view to finding out the mechanism(s) for observed effects.
Experiential Design: In vitro studies using isolated rat’s uteri.
Methods: Female albino rats (140-180g) pretreated 24 hours before experiments with diethylstilbestrol were sacrificed and the uterine horns carefully harvested into a beaker of De Jalon solution bubbled with 95% oxygen and 5% carbon dioxide. Each horn was mounted in an organ bath and allowed to equilibrate for 30 minutes, then the effects of graded doses of Acetylcholine, oxytocin and ELMO were established, using a physiograph and its accessories. The drugs were re-administered in the presence of their respective antagonists (Atropine for Acetylcholine and Atosiban for Oxytocin) and also in the presence of ELMO.
Results: Results obtained showed that while Acetylcholine and oxytocin induced uterine contractions, ELMO caused relaxation. ELMO significantly (P<0.05) blocked the uterine contractile effect of Acetylcholine but had no effect on Oxytocin induced uterine contractions. The experiments therefore indicate that ELMO contain active ingredients capable of inducing uterine relaxation via the muscarinic receptor pathway.
Conclusion: The extract therefore, may not be a valuable tocolytic agent in cases of Oxytocin induced uterine contractions, particularly in pregnancy but its observed strong anticholinergic activity may be exploited in the treatment of diseases associated with hyper activity of the parasympathetic arm of the autonomic nervous system.