Mitragyna speciosa is reported to be beneficial for the management of chronic pain and opioid withdrawal in the evolving opioid epidemic. Data on the blood-brain barrier (BBB) transport of mitragynine and 7-hydroxymitragynine, the active compounds of the plant, are still lacking and inconclusive. Here, we present for the first time the rate and the extent of mitragynine and 7-hydroxymitragynine transport across the BBB, with an investigation of their post-BBB intra-brain distribution. We utilized an in vitro BBB model to study the rate of BBB permeation of the compounds and their interaction with efflux transporter P-glycoprotein (P-gp). Mitragynine showed higher apical-to-basolateral (A-B, i.e. blood-to-brain side) permeability than 7-hydroxymitragynine. 7-Hydroxymitragynine showed a tendency to efflux, with efflux ratio (B-A/A-B) of 1.39. Both were found to inhibit the P-gp and are also subject to efflux by the P-gp. Assessment of the extent of BBB transport in vivo in rats from unbound brain to plasma concentration ratios (Kp,uu,brain ) revealed extensive efflux of both compounds, with less than 10 percent of unbound mitragynine and 7-hydroxymitragynine in plasma crossing the BBB. By contrast, the extent of intra-brain distribution was significantly different, with mitragynine having 18-fold higher brain tissue uptake in brain slice assay compared with 7-hydroxymitragynine. Mitragynine showed a moderate capacity to accumulate inside brain parenchymal cells, while 7-hydroxymitragynine showed restricted cellular barrier transport. The presented findings from this systematic investigation of brain pharmacokinetics of mitragynine and 7-hydroxymitragynine are essential for design and interpretation of in vivo experiments aiming to establish exposure-response relationship.
A specialized DNA extraction method and a SYBR Green quantitative polymerase chain reaction (SyG-qPCR) assay were combined to generate a ready-to-use kit for rapid detection of porcine admixtures in processed meat products. Our qPCR assay utilized repetitive LINE-1 elements specific to the genome of Sus scrofa domesticus (pig) as a target and incorporated internal controls. We improved the genomic DNA extraction method, and reduced extraction times to the minimum. The method was validated for specificity, sensitivity (0.001% w/w) and robustness, and values were compared with those of a commercially available kit. We also tested our method using 121 processed food products and consistently detected amplification only in samples containing pork. Due to its efficiency and cost-effectiveness, our method represents a valuable new method for detecting food adulteration with pork that is superior to existing quality control approaches.
Keeping in mind the pharmacological importance of 2-aminothiazole and 1,2,4-triazole heterocyclic moieties, a series of novel ethylated bi-heterocyclic acetamide hybrids, 9a-p, was synthesized in a multi-step protocol. The structures of newly synthesized compounds were characterized by 1H NMR, 13C NMR, IR and EI-MS spectral studies. The inhibitory effects of these bi-heterocyclic acetamides (9a-n) were evaluated against elastase and all these molecules were identified as potent inhibitors relative to the standard used. The Kinetics mechanism was analyzed by Lineweaver-Burk plots which revealed that, 9h, inhibited elastase competitively by forming an enzyme-inhibitor complex. The inhibition constants Ki calculated from Dixon plots for this compound was 0.9 µM. The computational study was articulate with the experimental results and these ligands unveiled good binding energy values (kcal/mol). So, these molecules can be considered as promising medicinal scaffolds for the treatment of skin melanoma, wrinkle formation, uneven pigmentation, and solar elastosis.
Aristolochic acid I (AAI) is a well-known genotoxic kidney carcinogen. Metabolic conversion of AAI into the DNA-reactive aristolactam-nitrenium ion is involved in the mode of action of tumor formation. This study aims to predict in vivo AAI-DNA adduct formation in the kidney of rat, mouse and human by translating the in vitro concentration-response curves for AAI-DNA adduct formation to the in vivo situation using physiologically based kinetic (PBK) modeling-based reverse dosimetry. DNA adduct formation in kidney proximal tubular LLC-PK1 cells exposed to AAI was quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry. Subsequently, the in vitro concentration-response curves were converted to predicted in vivo dose-response curves in rat, mouse and human kidney using PBK models. Results obtained revealed a dose-dependent increase in AAI-DNA adduct formation in the rat, mouse and human kidney and the predicted DNA adduct levels were generally within an order of magnitude compared with values reported in the literature. It is concluded that the combined in vitro PBK modeling approach provides a novel way to define in vivo dose-response curves for kidney DNA adduct formation in rat, mouse and human and contributes to the reduction, refinement and replacement of animal testing.
Edible bird's nest (EBN) is an expensive animal bioproduct due to its reputation as a food and delicacy with diverse medicinal properties. One kilogram of EBN costs ~$6000 in China. EBN and its products are consumed in mostly Asian countries such as China, Hong Kong, Taiwan, Singapore, Malaysia, Indonesia, Vietnam and Thailand, making up almost 1/3 of world population. The rapid growth in EBN consumption has led to a big rise in the trade scale of its global market. Presently, various fake materials such as tremella fungus, pork skin, karaya gum, fish swimming bladder, jelly, agar, monosodium glutamate and egg white are used to adulterate EBNs for earning extra profits. Adulterated or fake EBN may be hazardous to the consumers. Thus, it is necessary to identify of the adulterants. Several sophisticated techniques based on genetics, immunochemistry, spectroscopy, chromatography and gel electrophoresis have been used for the detection of various types of adulterants in EBN. This article describes the recent advances in the authentication methods for EBN. Different genetic, immunochemical, spectroscopic and analytical methods such as genetics (DNA) based techniques, enzyme-linked immunosorbent assays, Fourier transform infrared and Raman spectroscopic techniques, and chromatographic and gel electrophoretic methods have been discussed. Besides, significance of the reported methods that might pertain them to applications in EBN industry has been described. Finally, efforts have been made to discuss the challenges and future perspectives of the authentication methods for EBN.
α-Amylase is a target for type-2 diabetes mellitus treatment. However, small molecule inhibitors of α-amylase are currently scarce. In the course of developing small molecule α-amylase inhibitors, we designed and synthesized thiadiazole quinoline analogs (1-30), characterized by different spectroscopic techniques such as 1HNMR and EI-MS and screened for α-amylase inhibitory potential. Thirteen analogs 1, 2, 3, 4, 5, 6, 22, 23, 25, 26, 27, 28 and 30 showed outstanding α-amylase inhibitory potential with IC50 values ranges between 0.002±0.60 and 42.31±0.17μM which is many folds better than standard acarbose having IC50 value 53.02±0.12μM. Eleven analogs 7, 9, 10, 11, 12, 14, 15, 17, 18, 19 and 24 showed good to moderate inhibitory potential while seven analogs 8, 13, 16, 20, 21 and 29 were found inactive. Our study identifies novel series of potent α-amylase inhibitors for further investigation. Structure activity relationship has been established.
Coenzyme Q10 (CoQ10) is a vitamin-like oil-soluble molecule that has anti-oxidant and anti-ageing effects. To determine the most optimal CoQ10 delivery vehicle, CoQ10 was solubilised in both water and fish oil, and formulated into hydrogel, oleogel and bigel. Permeability of CoQ10 from each formulation across porcine ear skin was then evaluated. Furthermore, the effects of the omega-3 fatty eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids from fish oil on skin permeation were investigated by means of nuclear magnetic resonance (NMR) and computerised molecular modelling docking experiments. The highest drug permeation was achieved with the bigel formulation that proved to be the most effective vehicle in delivering CoQ10 across the skin membrane due to a combination of its adhesive, viscous and lipophilic properties. Furthermore, the interactions between CoQ10 and fatty acids revealed by NMR and molecular modelling experiments likely accounted for skin permeability of CoQ10. NMR data showed dose-dependent changes in proton chemical shifts in EPA and DHA. Molecular modelling revealed complex formation and large binding energies between fatty acids and CoQ10. This study advances the knowledge about bigels as drug delivery vehicles and highlights the use of NMR and molecular docking studies for the prediction of the influence of drug-excipient relationships at the molecular level.
Nipah virus (NiV) is a lethal bat-borne zoonotic virus that causes mild to acute respiratory distress and neurological manifestations in humans with a high mortality rate. NiV transmission to humans occurs via consumption of bat-contaminated fruit and date palm sap (DPS), or through direct contact with infected individuals and livestock. Since NiV outbreaks were first reported in pigs from Malaysia and Singapore, non-neutralizing antibodies against NiV attachment Glycoprotein (G) have also been detected in a few domestic mammals. NiV infection is initiated after NiV G binds to the host cell receptors Ephrin-B2 and Ephrin-B3. In this study, we assessed the degree of NiV host tropism in domestic and peridomestic mammals commonly found in Bangladesh that may be crucial in the transmission of NiV by serving as intermediate hosts. We carried out a protein-protein docking analysis of NiV G complexes (n = 52) with Ephrin-B2 and B3 of 13 domestic and peridomestic species using bioinformatics tools. Protein models were generated by homology modelling and the structures were validated for model quality. The different protein-protein complexes in this study were stable, and their binding affinity (ΔG) scores ranged between -8.0 to -19.1 kcal/mol. NiV Bangladesh (NiV-B) strain displayed stronger binding to Ephrin receptors, especially with Ephrin-B3 than the NiV Malaysia (NiV-M) strain, correlating with the observed higher pathogenicity of NiV-B strains. From the docking result, we found that Ephrin receptors of domestic rat (R. norvegicus) had a higher binding affinity for NiV G, suggesting greater susceptibility to NiV infections compared to other study species. Investigations for NiV exposure to domestic/peridomestic animals will help us knowing more the possible role of rats and other animals as intermediate hosts of NiV and would improve future NiV outbreak control and prevention in humans and domestic animals.
Edible seaweeds are valuable because of their organoleptic properties and complex polysaccharide content. A study was conducted to investigate the potential of dried edible seaweed extracts, its potential phenolic compounds and alginates for α-amylase inhibitory effects. The kinetics of inhibition was assessed in comparison with acarbose. The methanol extract of Laminaria digitata and the acetone extract of Undaria pinnatifida showed inhibitory activity against α-amylase, IC50 0.74 ± 0.02 mg/ml and 0.81 ± 0.03 mg/ml, respectively; both showed mixed-type inhibition. Phenolic compound, 2,5-dihydroxybenzoic acid was found to be a potent inhibitor of α-amylase with an IC50 value of 0.046 ± 0.004 mg/ml. Alginates found in brown seaweeds appeared to be potent inhibitors of α-amylase activity with an IC50 of (0.075 ± 0.010-0.103 ± 0.017) mg/ml, also a mixed-type inhibition. Overall, the findings provide information that crude extracts of brown edible seaweeds, phenolic compounds and alginates are potent α-amylase inhibitors, thereby potentially retarding glucose liberation from starches and alleviation of postprandial hyperglycaemia.
Plants that help in slowing down the digestion of triacylglycerols (TAGs) in the pancreas and small intestine of humans play an important role in the reduction of obesity. On the other hand, there may be plants or plant parts that stimulate intestinal lipolytic activity, thus contributing to greater TAG assimilation. The aim of this study was to evaluate the aqueous methanolic extracts of ninety eight (98) medicinal, herbal and aquatic plant materials from Malaysia for their effect on porcine pancreatic lipase (PPL) activity and to identify the structure of an anti-lipase compound from one of the sources. The degree of inhibition was also quantified as relative to orlistat activity against PPL (orlistat equivalents). Results revealed that while 19.4% of the extracts were found to have anti-lipase activity ≥80%, 12% were actually found to promote PPL activity. Twenty two percent (22.4%) exhibited moderate inhibition (41%-80%) and 2% were neutral toward PPL activity. The ripe fruit of Averrhoa carambola and the leaves of Archidendron jiringa (Jack) I.C Nielsen L. (jering), Cynometra cauliflora (nam-nam) and Aleurites moluccana (L.) Willd (candle nut/buah keras) had the highest (100%) anti-lipase activity and are equivalent to 0.11 µg orlistat/mL. Plants that stimulated lipase activity included Pimpinella anisum L. (aniseed/jintan manis), activating the enzyme by 186.5%. Kaempferol 3-O-rhamnoside was isolated from the ethyl acetate fraction of C. cauliflora leaves and found to be an active lipase inhibitor. The structure was elucidated using 1H-NMR, 13C-NMR and 2D-NMR analyses.
This study determined the risk factors and characteristics of vancomycin-resistant Enterococci (VRE) among individuals working with animals in Malaysia.
Porcine circovirus type 2 is the primary etiological agent associated with a group of complex multi-factorial diseases classified as Porcine Circovirus Associated Diseases (PCVAD). Sporadic cases reported in Malaysia in 2007 caused major economic losses to the 2.2 billion Malaysian ringgit (MYR) (approximately 0.7 billion US dollar) swine industry. The objective of the present study was to determine the association between the presence of PCV2 and occurrences of PCVAD.
Forty-seven Salmonella Typhimurium (33 zoonotic, 14 clinical) strains were tested for antimicrobial resistance using the standard disk diffusion method. Presence of relevant resistance genes and class 1 integrons were investigated by using PCR. Pulsed-field gel electrophoresis (PFGE) and plasmid profiling were carried out to determine the genomic diversity of Salmonella Typhimurium. Approximately 57.4% of S. Typhimurium were multidrug resistant (MDR) and showed high resistance rates to tetracycline (70.2%), sulphonamides (57.4%), streptomycin (53.1%), ampicillin (29.7%), nalidixic acid (27.6%), kanamycin (23.4%), chloramphenicol (21.2%) and trimethoprim (19.1%). Resistance towards cephalosporins was noted for cephalothin (27.6%), cephradine (21.2%), amoxicillin clavulanic acid (17.0%) and cephalexin (17.0%). Resistance genes, blaTEM, strA, aadA, sul1, sul2, tet(A), tet(B) and tet(C) were detected among the drug resistant strains. Thirty-three strains (70.2%) carried class 1 integrons, which were grouped in 9 different profiles. DNA sequencing identified sat, aadA, pse-1 and dfrA genes in variable regions on class 1 integrons. Thirty-five strains (74.4%) were subtyped to 22 different plasmid profiles, each with 1 - 6 plasmids (2.0 to 95 kb). PFGE subtyped the 47 strains into 39 profiles. In conclusion, high rates of multidrug-resistance were found among the Malaysian Salmonella Typhimurium strains. The emergence of multidrug-resistant Salmonella Typhimurium to cephalosporin antibiotics was also observed. The strains were very diverse and no persistent clone was observed. The emergence of MDR Salmonella Typhimurium is a worldwide problem and this report provides information for the better understanding of the prevalence and epidemiology of MDR S. Typhimurium in Malaysia.
Three ELISA test kits, the Randox ELISA beta-agonist test kit, Euro-Diagnostica test kit, and Ridascreen beta-agonist test kit, were evaluated for screening of meat and liver for beta-agonist residues in fortified and field-incurred samples. It was found that the Randox beta-agonist test kit was more suitable as a screening tool due to its accuracy, ease of use, and lower cost. The tests were able to detect beta-agonist residues at the minimum level of detection, as claimed by the suppliers. The performance of the method as assessed through recovery rates of beta-agonists in fortified samples was satisfactory with a low coefficient of variation (1-3%). Repeatability, as measured through the coefficient of correlation was also satisfactory. For field-incurred positive samples, the test kit showed a sensitivity of 100% and a low rate of false positives for goat and cow tissues. However, a high rate of apparent false positives was obtained for tissues of swine.
A random peptide library of heptamers displayed on the surface of M13 bacteriophage was used to identify specific epitopes of antibodies in pooled sera of swine naturally infected by Nipah virus. The selected heptapeptides were aligned with protein sequences of Nipah virus and several putative epitopes were identified within the nucleocapsid protein. A total of 41 of 60 (68%) selected phage clones had inserts resembling a region with the sequence SNRTQGE, located at the C-terminal end (amino acids 503-509) of the nucleocapsid protein. The binding of antibodies in the swine and human antisera to the phage clone was inhibited by a synthetic peptide corresponding to this region. Epitopes identified by phage display are consistent with the predicted antigenic sites for the Nipah virus nucleocapsid protein. The selected phage clone used as a coating antigen discriminated swine and human Nipah virus sera-positive from sera-negative samples exhibiting characteristics, which might be attractive for diagnostic tests.
BACKGROUND: Between September 1998 and June 1999, there was an outbreak of severe viral encephalitis due to Nipah virus, a newly discovered paramyxovirus, in Malaysia.
METHODS: We studied the clinical features of the patients with Nipah virus encephalitis who were admitted to a medical center in Kuala Lumpur. The case definition was based on epidemiologic, clinical, cerebrospinal fluid, and neuroimaging findings.
RESULTS: Ninety-four patients with Nipah virus infection were seen from February to June 1999 (mean age, 37 years; ratio of male patients to female patients, 4.5 to 1). Ninety-three percent had had direct contact with pigs, usually in the two weeks before the onset of illness, suggesting that there was direct viral transmission from pigs to humans and a short incubation period. The main presenting features were fever, headache, dizziness, and vomiting. Fifty-two patients (55 percent) had a reduced level of consciousness and prominent brain-stem dysfunction. Distinctive clinical signs included segmental myoclonus, areflexia and hypotonia, hypertension, and tachycardia and thus suggest the involvement of the brain stem and the upper cervical spinal cord. The initial cerebrospinal fluid findings were abnormal in 75 percent of patients. Antibodies against Hendra virus were detected in serum or cerebrospinal fluid in 76 percent of 83 patients tested. Thirty patients (32 percent) died after rapid deterioration in their condition. An abnormal doll's-eye reflex and tachycardia were factors associated with a poor prognosis. Death was probably due to severe brain-stem involvement. Neurologic relapse occurred after initially mild disease in three patients. Fifty patients (53 percent) recovered fully, and 14 (15 percent) had persistent neurologic deficits.
CONCLUSIONS: Nipah virus causes a severe, rapidly progressive encephalitis with a high mortality rate and features that suggest involvement of the brain stem. The infection is associated with recent contact with pigs.
The Nipah virus was discovered twenty years ago, and there is considerable information available regarding the specificities surrounding this virus such as transmission, pathogenesis and genome. Belonging to the Henipavirus genus, this virus can cause fever, encephalitis and respiratory disorders. The first cases were reported in Malaysia and Singapore in 1998, when affected individuals presented with severe febrile encephalitis. Since then, much has been identified about this virus. These single-stranded RNA viruses gain entry into target cells via a process known as macropinocytosis. The viral genome is released into the cell cytoplasm via a cascade of processes that involves conformational changes in G and F proteins which allow for attachment of the viral membrane to the cell membrane. In addition to this, the natural reservoirs of this virus have been identified to be fruit bats from the genus Pteropus. Five of the 14 species of bats in Malaysia have been identified as carriers, and this virus affects horses, cats, dogs, pigs and humans. Various mechanisms of transmission have been proposed such as contamination of date palm saps by bat feces and saliva, nosocomial and human-to-human transmissions. Physical contact was identified as the strongest risk factor for developing an infection in the 2004 Faridpur outbreak. Geographically, the virus seems to favor the Indian sub-continent, Indonesia, Southeast Asia, Pakistan, southern China, northern Australia and the Philippines, as demonstrated by the multiple outbreaks in 2001, 2004, 2007, 2012 in Bangladesh, India and Pakistan as well as the initial outbreaks in Malaysia and Singapore. Multiple routes of the viremic spread in the human body have been identified such as the central nervous system (CNS) and respiratory system, while virus levels in the body remain low, detection in the cerebrospinal fluid is comparatively high. The virus follows an incubation period of 4 days to 2 weeks which is followed by the development of symptoms. The primary clinical signs include fever, headache, vomiting and dizziness, while the characteristic symptoms consist of segmental myoclonus, tachycardia, areflexia, hypotonia, abnormal pupillary reflexes and hypertension. The serum neutralization test (SNT) is the gold standard of diagnosis followed by ELISA if SNT cannot be carried out. On the other hand, treatment is supportive since there a lack of effective pharmacological therapy and only one equine vaccine is currently licensed for use. Prevention of outbreaks seems to be a more viable approach until specific therapeutic strategies are devised.
Nipah virus is a recently emergent paramyxovirus that is capable of causing severe disease in both humans and animals. The first outbreak of Nipah virus occurred in Malaysia and Singapore in 1999 and, more recently, outbreaks were detected in Bangladesh. In humans, Nipah virus causes febrile encephalitis with respiratory syndrome that has a high mortality rate. The reservoir for Nipah virus is believed to be fruit bats, and humans are infected by contact with infected bats or by contact with an intermediate animal host such as pigs. Person to person spread of the virus has also been described. Nipah virus retains many of the genetic and biologic properties found in other paramyxoviruses, though it also has several unique characteristics. However, the virologic characteristics that allow the virus to cause severe disease over a broad host range, and the epidemiologic, environmental and virologic features that favor transmission to humans are unknown. This review summarizes what is known about the virology, epidemiology, pathology, diagnosis and control of this novel pathogen.
Dutasteride, used for treating benign prostate hyperplasia (BPH), promotes hair growth. To enhance delivery to the hair follicles and reduce systemic effects, in this study dutasteride has been formulated for topical application, in a nanostructured lipid carrier (NLC) coated with chitosan oligomer-stearic acid (CSO-SA). CSO-SA has been successfully synthesized, as confirmed using1H NMR and FTIR. Formulation of dutasteride-loaded nanostructured lipid carriers (DST-NLCs) was optimized using a 23full factorial design. This formulation was coated with different concentrations of stearic acid-chitosan solution. Coating DST-NLCs with 5% SA-CSO increased mean size from 187.6±7.0nm to 220.1±11.9nm, and modified surface charge, with zeta potentials being -18.3±0.9mV and +25.8±1.1mV for uncoated and coated DST-NLCs respectively. Transmission electron microscopy showed all formulations comprised approximately spherical particles. DST-NLCs, coated and uncoated with CSO-SA, exhibited particle size stability over 60days, when stored at 4-8°C. However, NLCs coated with CSO (without conjugation) showed aggregation when stored at 4-8°C after 30days. The measured particle size for all formulations stored at 25°C suggested aggregation, which was greatest for DST-NLCs coated with 10% CSO-SA and 5% CSO. All nanoparticle formulations exhibited rapid release in an in vitro release study, with uncoated NLCs exhibiting the fastest release rate. Using a Franz diffusion cell, no dutasteride permeated through pig ear skin after 48h, such that it was not detected in the receptor chamber for all samples. The amount of dutasteride in the skin was significantly different (p<0.05) for DST-NLCs (6.09±1.09μg/cm2) without coating and those coated with 5% CSO-SA (2.82±0.40μg/cm2), 10% CSO-SA (2.70±0.35μg/cm2) and CSO (2.11±0.64μg/cm2). There was a significant difference (p<0.05) in the cytotoxicity (IC50) between dutasteride alone and in the nanoparticles. DST-NLCs coated and uncoated with CSO-SA increased the maximum non-toxic concentration by 20-fold compared to dutasteride alone. These studies indicate that a stearic acid-chitosan conjugate was successfully prepared, and modified the surface charge of DST-NLCs from negative to positive. These stable, less cytotoxic, positively-charged dutasteride-loaded nanostructured lipid carriers, with stearic acid-chitosan oligomer conjugate, are appropriate for topical delivery and have potential for promotion of hair growth.