Ridwan Bin Rashid

Pharmacological Activities of Blumea lacera (Burm. f) DC: A Medicinal Plant of Bangladesh

Aims: The crude methanol extract of whole plant of Blumea lacera (Burn.f.) DC. has been investigated for anti-diarrheal, antimicrobial, anxiolytic, anti-atherothrombosis, membrane stabilizing and alpha-amylase inhibitory activities. Place and Duration of Study: The study was carried out in 2013 in the Department of Pharmacy, Southern University Bangladesh, Chittagong, Bangladesh. Methodology: Test for anti-diarrheal activity was carried out by castor oil-induced diarrhea in mice. The preliminary antimicrobial activity was determined by the agar disc diffusion method. The anxiolytic activity was examined in mice by using the hole board test and open field test (OFT). The anti-atherothrombosis activity was evaluated using standard streptokinase. The membrane stabilizing activity was assessed by using hypotonic solution induced hemolysis of human erythrocyte. The plant extract was also assessed for anti-diabetic ability using In vitro α-amylase inhibitory potential. The αamylase inhibitory activity of B. lacera was measured using the starch-iodine method.

Theoretically Guided Analytical Method Development and Validation for the Estimation of Rifampicin in a Mixture of Isoniazid and Pyrazinamide by UV Spectrophotometer

A simple, rapid, economic, accurate, and precise method for the estimation of rifampicin in a mixture of isoniazid and pyrazinamide by UV spectrophotometeric technique (guided by the theoretical investigation of physicochemical properties) was developed and validated. Theoretical investigations revealed that isoniazid and pyrazinamide both were freely soluble in water and slightly soluble in ethyl acetate whereas rifampicin was practically insoluble in water but freely soluble in ethyl acetate. This indicates that ethyl acetate is an effective solvent for the extraction of rifampicin from a water mixture of isoniazid and pyrazinamide. Computational study indicated that pH range of 6.0–8.0 would favor the extraction of rifampicin. Rifampicin is separated from isoniazid and pyrazinamide at pH 7.4 ± 0.1 by extracting with ethyl acetate. The ethyl acetate was then analyzed at λmax of 344.0 nm. The developed method was validated for linearity, accuracy and precision according to ICH guidelines. The proposed method exhibited good linearity over the concentration range of 2.5–35.0 μg/mL. The intraday and inter-day precision in terms of % RSD ranged from 1.09 to 1.70% and 1.63 to 2.99%, respectively. The accuracy (in terms of recovery) of the method varied from of 96.7 ± 0.9 to 101.1 ± 0.4%. The LOD and LOQ were found to be 0.83 and 2.52 μg/mL, respectively. In addition, the developed method was successfully applied to determine rifampicin combination (isoniazid and pyrazinamide) brands available in Bangladesh.

Polymethoxyflavones from Nicotiana plumbaginifolia (Solanaceae) Exert Antinociceptive and Neuropharmacological Effects in Mice

Polymethoxylavones (PMFs) are known to exhibit significant anti-inflammatory and neuroprotective properties. Nicotiana plumbaginifolia, an annual Bangladeshi herb, is rich in polymethoxyflavones that possess significant analgesic and anxiolytic activities. The present study aimed to determine the antinociceptive and neuropharmacological activities of polyoxygenated flavonoids namely- 3,3′,5,6,7,8-hexamethoxy-4′,5′-methylenedioxyflavone (1), 3,3′,4′,5′,5,6,7,8-octamethoxyflavone (exoticin) (2), 6,7,4′,5′-dimethylenedioxy-3,5,3′-trimethoxyflavone (3), and 3,3′,4′,5,5′,8-hexamethoxy-6,7-methylenedioxyflavone (4), isolated and identified from N. plumbaginifolia. Antinociceptive activity was assessed using the acetic-acid induced writhing, hot plate, tail immersion, formalin and carrageenan-induced paw edema tests, whereas neuropharmacological effects were evaluated in the hole cross, open field and elevated plus maze test. Oral treatment of compounds 1, 3, and 4 (12.5–25 mg/kg b.w.) exhibited dose-dependent and significant (p < 0.01) antinociceptive activity in the acetic-acid, formalin, carrageenan, and thermal (hot plate)-induced pain models. The association of ATP-sensitive K+ channel and opioid systems in their antinociceptive effect was obvious from the antagonist effect of glibenclamide and naloxone, respectively. These findings suggested central and peripheral antinociceptive activities of the compounds. Compound 1, 3, and 4 (12.5 mg/kg b.w.) demonstrated significant (p < 0.05) anxiolytic-like activity in the elevated plus-maze test, while the involvement of GABAA receptor in the action of compound 3 and 4 was evident from the reversal effects of flumazenil. In addition, compounds 1 and 4 (12.5–25 mg/kg b.w) exhibited anxiolytic activity without altering the locomotor responses. The present study suggested that the polymethoxyflavones (1–4) from N. Plumbaginifolia could be considered as suitable candidates for the development of analgesic and anxiolytic agents.

A Comparative Analysis of Vibrio cholerae Contamination in Point-of-Drinking and Source Water in a Low-Income Urban Community, Bangladesh

Bangladesh is a cholera endemic country with a population at high risk of cholera. Toxigenic and non-toxigenic Vibrio cholerae (V. cholerae) can cause cholera and cholera-like diarrheal illness and outbreaks. Drinking water is one of the primary routes of cholera transmission in Bangladesh. The aim of this study was to conduct a comparative assessment of the presence of V. cholerae between point-of-drinking water and source water, and to investigate the variability of virulence profile using molecular methods of a densely populated low-income settlement of Dhaka, Bangladesh. Water samples were collected and tested for V. cholerae from “point-of-drinking” and “source” in 477 study households in routine visits at 6 week intervals over a period of 14 months. We studied the virulence profiles of V. cholerae positive water samples using 22 different virulence gene markers present in toxigenic O1/O139 and non-O1/O139 V. cholerae using polymerase chain reaction (PCR). A total of 1,463 water samples were collected, with 1,082 samples from point-of-drinking water in 388 households and 381 samples from 66 water sources. V. cholerae was detected in 10% of point-of-drinking water samples and in 9% of source water samples. Twenty-three percent of households and 38% of the sources were positive for V. cholerae in at least one visit. Samples collected from point-of-drinking and linked sources in a 7 day interval showed significantly higher odds (P < 0.05) of V. cholerae presence in point-of-drinking compared to source [OR = 17.24 (95% CI = 7.14–42.89)] water. Based on the 7 day interval data, 53% (17/32) of source water samples were negative for V. cholerae while linked point-of-drinking water samples were positive. There were significantly higher odds (p < 0.05) of the presence of V. cholerae O1 [OR = 9.13 (95% CI = 2.85–29.26)] and V. cholerae O139 [OR = 4.73 (95% CI = 1.19–18.79)] in source water samples than in point-of-drinking water samples. Contamination of water at the point-of-drinking is less likely to depend on the contamination at the water source. Hygiene education interventions and programs should focus and emphasize on water at the point-of-drinking, including repeated cleaning of drinking vessels, which is of paramount importance in preventing cholera.

Development and Validation of a Novel Real-time Assay for the Detection and Quantification of Vibrio cholerae

Vibrio cholerae O1 and O139 has been known for its ability to cause epidemics. These strains produce cholera toxin which is the main cause of secretory diarrhea. V. cholerae non-O1 and non-O139 strains are also capable of causing gastroenteritis as well as septicemia and peritonitis. It has been proven that virulence factors such as T6SS, hapA, rtxA, and hlyA are present in almost all V. cholerae strains. It is imperative that viable but non-culturable cells of V. cholerae are also detected since they are also known to cause diarrhea. Thus, the aim of this study was to develop an assay that detects all V. cholerae regardless of their serotype, culturable state, and virulence genes present, by targeting the species specific conserved ompW sequence. The developed assay meets these goals with 100% specificity and is capable of detecting as low as 5.46 copy number of V. cholerae. Detection is rapid since neither lengthy incubation period nor electrophoresis is required. The assay had excellent repeatability (CV%: 0.24–1.32) and remarkable reproducibility (CV%: 1.08–3.7). Amplification efficiencies in the 89–100% range were observed. The assay is more economical than Taqman-based multiplex real-time PCR assays. Compared to other real-time assays, the ompW assay is specific and sensitive, has better repeatability and reproducibility, and is more economical.

Genotypic Analysis of the Virulence and Antibiotic Resistance Genes in Campylobacter species in silico .

Campylobacter species is responsible for 400-500 million diarrhea cases worldwide every year. Emergence of antibiotic resistance has further complicated the scenario. A wide range of virulence factors and resistance genes are present in Campylobacter species and it is hypothesized there are genotypic variations in the prevalence of these genes. The study was conducted to investigate the presence of virulence and antibiotic resistance genes as well as to investigate difference in prevalence rate based on genotype through in silico tools. Among 26 species studied, sixteen isolates (61.54%) had the cdtB gene that breaks the double helix bonds. The cdtA genes were detected in ten (38.46%) C. jejuni strains while fifty percent (n=13) isolates harbored the cdtC genes. Ten isolates that harboured all three adjacent cdt genes were most toxigenic. The lipo-oligosaccharides associated genes, cgtB and wlaN, responsible for β-1,3 galactosyltransferase production, were found in 7.69% and 30.77% of the isolates, respectively. About 57.69% isolates expressed waaC genes. Invasion protein ciaB, outer membrane phospholipase A pldA and IV secretory protein virB11 were found in 53.85%, 34.62% and 7.69% of the isolates, respectively. Six isolates (23.08%) expressed both tetO and tetA genes while one isolate expressed only tetA resistance gene. Seven isolates (26.92%) had changes in gyrB genes that conferred the fluoroquinolone resistance. In silico PFGE typing found that genotype 3 contained all the virulence genes except cgtB gene while genotype 3 and 4 contained mutated gyrB gene. Genotype 1 and 5 contained no virulence and antibiotic resistance genes. Our data helps to predict the possibility of the presence of virulence and antibiotic resistance genes and helps to select appropriate antibiotic that are more efficacious.

Phylogenetic Analysis of the Antibiotic Resistance Genes in Salmonella Species in silico .

Antibiotic resistance is an emerging problem in both developed and developing countries. It has been responsible for 700,000 deaths worldwide. Some genotypes of bacteria are sensitive to certain antibiotics than others. Hence by conducting phylogenetic analysis of bacteria and detecting the presence of resistance genes in each genotype, we can select the antibiotic that would be most effective for the bacteria in that certain genotype. A total of forty-five Salmonella species were investigated for the presence of antibiotic resistance genes through in silico PCR (polymerase chain reaction) amplification and PFGE (pulsed-field gel electrophoresis) analysis was conducted to assess the phylogenetic relationship. Total twenty-eight antibiotic resistance genes were selected for screening the isolates and seventeen antibiotic resistance genes among the Salmonella strains were found. Almost all the isolates (n=43) exhibited PCR amplification product for gyrA genes while fluoroquinolone resistance gyrB (66.67%), parC (68.89%) and parE (15.56%) genes were also present. About 15.56% and 11.11% isolates were found to harbor adenylyltransferase gene, aadA1 and aadA2, respectively while phosphotransferase gene was detected in only one isolate. Two isolates expressed both chloramphenicol acetyltransferase genes, cat1 and cat2. Three isolates (6.67%) harbored chloramphenicol resistance gene cmlA gene while two isolates (4.44%) expressed florfenicol resistance gene, floR. Tetracycline resistance gene, tetA was more prevalent (8.89%) than tetG genes (2.22%). Salmonella harbored all three sulfonamide resistance genes while sulII was more prevalent (17.78%). Genotype 2 contained fifteen antibiotic resistance genes while genotype 3 contained only one antibiotic resistance genes. These investigations used a computer aided approach to genotype isolates and assess the difference in antibiotic resistance profile of Salmonella species based on genotype. This data helps to predict antibiotic resistance genes that might be present for an isolate of known genotype and select antibiotic for the treatment of Salmonella infections based on their phylogenetic group.

Preliminary Phytochemical Screenings and Antipyretic, Analgesic and Anti-inflammatory Activities of Methanol Extract of Vernonia cinerea Less. (Fam: Asteraceae)

Aims: The aim of the current study was to undertake phytochemical screenings and evaluate antipyretic, analgesic and anti-inflammatory activities of the methanol extract of whole plant of Vernonia cinerea Less. (VCME). Place and Duration of Study: The study was carried out for one year in 2012 in the Department of Pharmacy, Southern University Bangladesh, Chittagong, Bangladesh. Methodology: For preliminary phytochemical screenings, the crude methanol extract of V. cinerea was subjected to various tests to determine the chemical nature of the extract. Antipyretic activity was assessed by the yeast-induced hyperthermia in mice. The analgesic property was evaluated by formalin-induced writhing test. Acetyl salicylic acid