Kang Nee Ting

Molecular and pharmacological evidence for MT1 melatonin receptor subtype in the tail artery of juvenile Wistar rats

In this study reverse transcriptase‐polymerase chain reaction (RT–PCR) has been used to identify mt1 and MT2 receptor mRNA expression in the rat tail artery. The contributions of both receptors to the functional response to melatonin were examined with the putative selective MT2 receptor antagonists, 4‐phenyl‐2‐propionamidotetraline (4‐P‐PDOT) and 2‐benzyl‐N‐pentanoyltryptamine. In addition, the action of melatonin on the second messenger cyclic AMP was investigated. Using RT–PCR, mt1 receptor mRNA was detected in the tail artery from seven rats. In contrast MT2 receptor mRNA was not detected even after nested PCR. At low concentrations of the MT2 selective ligands, neither 10 nM 4‐P‐PDOT (pEC50=8.70±0.31 (control) vs 8.73±0.16, n=6) nor 60 nM 2‐benzyl‐N‐pentanoyltryptamine (pEC50=8.53±0.20 (control) vs 8.83±0.38, n=6) significantly altered the potency of melatonin in the rat tail artery. At concentrations non‐selective for mt1 and MT2 receptors, 4‐P‐PDOT (3 μM) and 2‐benzyl‐N‐pentanoyltryptamine (5 μM) caused a significant rightward displacement of the vasoconstrictor effect of melatonin. In the case of 4‐P‐PDOT, the estimated pKB (6.17±0.16, n=8) is similar to the binding affinity for mt1 receptor. Pre‐incubation with 1 μM melatonin did not affect the conversion of [3H]‐adenine to [3H]‐cyclic AMP under basal condition (0.95±0.19% conversion (control) vs 0.92±0.19%, n=4) or following exposure to 30 μM forskolin (5.20±1.30% conversion (control) vs 5.35±0.90%, n=4). Based on the above findings, we conclude that melatonin receptor on the tail artery belongs to the MT1 receptor subtype, and that this receptor is probably independent of the adenylyl cyclase pathway.

Optimal methods for evaluating antimicrobial activities from plant extracts.

The search for antimicrobial agents from plants has been a growing interest in the last few decades. However, results generated from many of these studies cannot be directly compared due to the absence of standardization in particular antimicrobial methods employed. The need for established methods with consistent results for the evaluation of antimicrobial activities from plant extracts has been proposed by many researchers. Nevertheless, there are still many studies reported in the literature describing different methodologies. The aim of this study was to find optimal methods to give consistent quantitative antimicrobial results for studying plant extracts. Three different agar-based assays (pour plate disc diffusion (PPDD), streak plate disc diffusion (SPDD) and well-in agar (WA)) and one broth-based (turbidometric (TB)) assay were used in this study. Extracts from two plant species (Duabanga grandiflora and Acalypha wilkesiana) were tested on two bacterial species, namely Escherichia coli and Staphylococcus aureus. Amongst the agar-based assays, PPDD produced the most reproducible results. TB was able to show the inhibitory effects of the test samples on the growth kinetic of the bacteria including plant extracts with low polarity. We propose that both agar- (i.e PPDD) and broth-based assays should be employed when assessing the antimicrobial activity of plant crude extracts.

High Correlation of 2,2-diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging, Ferric Reducing Activity Potential and Total Phenolics Content Indicates Redundancy in Use of All Three Assays to Screen for Antioxidant Activity of Extracts of Plants from the Malaysian Rainforest

Extracts of plants from the Malaysian rainforest and other fragile habitats are being researched intensively for identification of beneficial biological actions, with assessment of antioxidant behavior being a common component of such assessments. A number of tests for antioxidant behavior are used, with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reduction activity potential (FRAP) assays often being used in parallel, and also with measurement of total phenolics content (TPC) as a surrogate marker for antioxidant capacity. The present study investigated the possible redundancy in using all three assays to determine antioxidant capacity in 92 extracts obtained from 27 plants from the Malaysian rainforest. The results demonstrated that the assays displayed a high (R ≥ 0.82) and significant (P < 0.0001) correlation with one another, indicating a high level of redundancy if all three assays are used in parallel. This appears to be a waste of potentially valuable plant extracts. Because of problems with the FRAP assay relating to color interference and variable rates of reaction point, the DPPH assay is the preferred assay in preliminary screening of extracts of plants from the Malaysian rainforest.

Acalypha wilkesiana extracts induce apoptosis by causing single strand and double strand DNA breaks

ETHNOPHARMACOLOGICAL RELEVANCE The seeds of Acalypha wilkesiana have been used empirically by traditional healers in Southwest Nigeria together with other plants as a powder mixture to treat patients with breast tumours and inflammation. AIM OF THE STUDY There is an increasing interest among researchers in searching for new anticancer drugs from natural resources, particularly plants. This study aimed to investigate the anticancer properties of Acalypha wilkesiana extracts and the characteristics of DNA damage against brain and lung cancer cells. MATERIALS AND METHODS The antiproliferative activity of Acalypha wilkesiana extracts (ethyl acetate, hexane, and ethanol) was examined on human glioma (U87MG), human lung carcinoma (A549), and human lung fibroblast (MRC5) cells. RESULTS Cell viability MTT assay revealed that ethyl acetate extract of the plant possessed significant antiproliferative effects against both U87MG (GI(50)=28.03 ± 6.44 μg/ml) and A549 (GI(50)=89.63 ± 2.12 μg/ml) cells (p value<0.0001). The hexane extract was found to exhibit crucial antiproliferative effects on U87MG (GI(50)=166.30 ± 30.50 μg/ml) (p value<0.0001) but not on A549 cells. Neither plant extract possessed noticeable antiproliferative effects on the non-cancerous MRC5 cells (GI(50)>300 μg/ml). The ethanol extract showed no antiproliferative effects on any cell line examined. Haematoxylin & Eosin (H & E) staining and single cell gel electrophoresis (SCGE) comet assay confirmed that plant extract-treated cells underwent apoptosis and not necrosis. SCGE comet assays confirmed that plant extracts caused both single strand (SSB) and double strand (DSB) DNA breaks that led to the execution of apoptosis. CONCLUSION The extracts (especially ethyl acetate and hexane) of Acalypha wilkesiana possess valuable cytotoxic effects that trigger apoptosis in U87MG and A549 cancer cells through induction of DNA SSBs and DSBs.

Community pharmacists’ views on adverse drug reactions reporting in Malaysia: a pilot study

Objectives To investigate community pharmacists’ knowledge, attitudes and views on adverse drug reaction (ADR) reporting. Setting Seven community pharmacies in Malaysia. Method Structured interviews with community pharmacists. Informed consent was obtained and interviews were audio-recorded and transcribed verbatim. Main Outcome Measures Content analysis of themes on awareness of ADR reporting, reporting activities, attitudes and views on patient reporting. Results All pharmacists claimed to have some knowledge of a reporting system but only one had submitted a report directly to the regulatory authority. Despite the low level of reporting activities, all participants agreed that it was part of their professional obligation to report an ADR. Most participants were not aware of the direct patient reporting scheme and were skeptical about its success. Lack of awareness and patients’ limited knowledge about their medications were viewed as barriers to patient reporting. Local attitudinal issues including pharmacists’ attitude towards ADR reporting were described as possible contributing factors. Conclusion Community pharmacists have an important role in reporting ADRs. Many Malaysian patients are still perceived to be ill-informed of their medications, an important determinant to the success of patient reporting. There is a need for further training about ADRs and ADR reporting for health professionals and further education for patients.

Cell Wall Perturbation Sensitizes Fungi to the Antimalarial Drug Chloroquine

ABSTRACT Chloroquine (CQ) has been a mainstay of antimalarial drug treatment for several decades. Additional therapeutic actions of CQ have been described, including some reports of fungal inhibition. Here we investigated the action of CQ in fungi, including the yeast model Saccharomyces cerevisiae. A genomewide yeast deletion strain collection was screened against CQ, revealing that bck1Δ and slt2Δ mutants of the cell wall integrity pathway are CQ hypersensitive. This phenotype was rescued with sorbitol, consistent with cell wall involvement. The cell wall-targeting agent caffeine caused hypersensitivity to CQ, as did cell wall perturbation by sonication. The phenotypes were not caused by CQ-induced changes to cell wall components. Instead, CQ accumulated to higher levels in cells with perturbed cell walls: CQ uptake was 2- to 3-fold greater in bck1Δ and slt2Δ mutants than in wild-type yeast. CQ toxicity was synergistic with that of the major cell wall-targeting antifungal drug, caspofungin. The MIC of caspofungin against the yeast pathogen Candida albicans was decreased 2-fold by 250 μM CQ and up to 8-fold at higher CQ concentrations. Similar effects were seen in Candida glabrata and Aspergillus fumigatus. The results show that the cell wall is critical for CQ resistance in fungi and suggest that combination treatments with cell wall-targeting drugs could have potential for antifungal treatment.

Cytotoxicity and apoptotic activities of alpha-, gamma- and delta-tocotrienol isomers on human cancer cells.

BackgroundTocotrienols, especially the gamma isomer was discovered to possess cytotoxic effects associated with the induction of apoptosis in numerous cancers. Individual tocotrienol isomers are believed to induce dissimilar apoptotic mechanisms in different cancer types. This study was aimed to compare the cytotoxic potency of alpha-, gamma- and delta-tocotrienols, and to explore their resultant apoptotic mechanisms in human lung adenocarcinoma A549 and glioblastoma U87MG cells which are scarcely researched.MethodsThe cytotoxic effects of alpha-, gamma- and delta-tocotrienols in both A549 and U87MG cancer cells were first determined at the cell viability and morphological aspects. DNA damage types were then identified by comet assay and flow cytometric study was carried out to support the incidence of apoptosis. The involvements of caspase-8, Bid, Bax and mitochondrial membrane permeability (MMP) in the execution of apoptosis were further expounded.ResultsAll tocotrienols inhibited the growth of A549 and U87MG cancer cells in a concentration- and time-dependent manner. These treated cancer cells demonstrated some hallmarks of apoptotic morphologies, apoptosis was further confirmed by cell accumulation at the pre-G1 stage. All tocotrienols induced only double strand DNA breaks (DSBs) and no single strand DNA breaks (SSBs) in both treated cancer cells. Activation of caspase-8 leading to increased levels of Bid and Bax as well as cytochrome c release attributed by the disruption of mitochondrial membrane permeability in both A549 and U87MG cells were evident.ConclusionsThis study has shown that delta-tocotrienol, in all experimental approaches, possessed a higher efficacy (shorter induction period) and effectiveness (higher induction rate) in the execution of apoptosis in both A549 and U87MG cancer cells as compared to alpha- and gamma-tocotrienols. Tocotrienols in particular the delta isomer can be an alternative chemotherapeutic agent for treating lung and brain cancers.

Criofolinine and vernavosine, new pentacyclic indole alkaloids incorporating pyrroloazepine and pyridopyrimidine moieties derived from a common yohimbine precursor

Two new indole alkaloids characterized by previously unencountered natural product skeletons, viz., criofolinine (1), incorporating a pyrroloazepine motif within a pentacyclic ring system, and vernavosine (2, isolated as its ethyl ether derivative 3, which on hydrolysis regenerated the putative precursor alkaloid 2), incorporating a pyridopyrimidine moiety embedded within a pentacyclic carbon framework, were isolated from a Malayan Tabernaemontana species. The structures and absolute configuration of these alkaloids were determined on the basis of NMR and MS analysis and confirmed by X-ray diffraction analysis.

Reversal of Ampicillin Resistance in MRSA via Inhibition of Penicillin-Binding Protein 2a by Acalypha wilkesiana

The inhibitory activity of a semipure fraction from the plant, Acalypha wilkesiana assigned as 9EA-FC-B, alone and in combination with ampicillin, was studied against methicillin-resistant Staphylococcus aureus (MRSA). In addition, effects of the combination treatment on PBP2a expression were investigated. Microdilution assay was used to determine the minimal inhibitory concentrations (MIC). Synergistic effects of 9EA-FC-B with ampicillin were determined using the fractional inhibitory concentration (FIC) index and kinetic growth curve assay. Western blot experiments were carried out to study the PBP2a expression in treated MRSA cultures. The results showed a synergistic effect between ampicillin and 9EA-FC-B treatment with the lowest FIC index of 0.19 (synergism ≤ 0.5). The presence of 9EA-FC-B reduced the MIC of ampicillin from 50 to 1.56 μg mL−1. When ampicillin and 9EA-FC-B were combined at subinhibitory level, the kinetic growth curves were suppressed. The antibacterial effect of 9EA-FC-B and ampicillin was shown to be synergistic. The synergism is due the ability of 9EA-FC-B to suppress the activity of PBP2a, thus restoring the susceptibility of MRSA to ampicillin. Corilagin was postulated to be the constituent responsible for the synergistic activity showed by 9EA-FC-B.

Quinine interactions with tryptophan and tyrosine in malaria patients, and implications for quinine responses in the clinical setting.

OBJECTIVES Recent work with the yeast model revealed that the antiprotozoal drug quinine competes with tryptophan for uptake via a common transport protein, causing cellular tryptophan starvation. In the present work, it was hypothesized that similar interactions may occur in malaria patients receiving quinine therapy. PATIENTS AND METHODS A direct observational study was conducted in which plasma levels of drug and amino acids (tryptophan, tyrosine and phenylalanine) were monitored during quinine treatment of malaria patients with Plasmodium falciparum infections. RESULTS Consistent with competition for uptake from plasma into cells, plasma tryptophan and tyrosine levels increased ≥2-fold during quinine therapy. Plasma quinine levels in individual plasma samples were significantly and positively correlated with tryptophan and tyrosine in the same samples. Control studies indicated no effect on phenylalanine. Chloroquine treatment of Plasmodium vivax-infected patients did not affect plasma tryptophan or tyrosine. During quinine treatment, plasma tryptophan was significantly lower (and quinine significantly higher) in patients experiencing adverse drug reactions. CONCLUSIONS Plasma quinine levels during therapy are related to patient tryptophan and tyrosine levels, and these interactions can determine patient responses to quinine. The study also highlights the potential for extrapolating insights directly from the yeast model to human malaria patients.