We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 μM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 μM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors.
Anthelmintic resistance is widespread in gastrointestinal nematode populations, such that there is a consistent need to search for new anthelmintics. However, the cost of screening for new compounds is high and has a very low success rate. Using the knowledge of traditional healers from Borneo Rainforests (Sarawak, Malaysia), we have previously shown that some traditional medicinal plants are a rich source of potential new anthelmintic drug candidates. In this study, Picria fel-terrae Lour. plant extract, which has previously shown promising anthelmintic activities, was fractionated via the use of a solid phase extraction cartridge and each isolated fraction was then tested on free-living nematode Caenorhabditis elegans and the parasitic nematode Haemonchus contortus. We found that a single fraction was enriched for nematocidal activity, killing ≥90% of C. elegans adults and inhibiting the motility of exsheathed L3 of H. contortus, while having minimal cytotoxic activity in mammalian cell culture. Metabolic profiling and chemometric analysis of the effective fraction indicated medium chained fatty acids and phenolic acids were highly represented.
Piper Betle L. (PB) belongs to the Piperaceae family. The presence of a fairly large quantity of diastase in the betel leaf is deemed to play an important role in starch digestion and calls for the study of weight loss activities and metabolite profile from PB leaf extracts using metabolomics approach to be performed. PB dried leaves were extracted with 70% ethanol and the extracts were subjected to five groups of rats fed with high fat (HF) and standard diet (SD). They were then fed with the extracts in two doses and compared with a negative control group given water only according to the study protocol. The body weights and food intakes were monitored every week. At the end of the study, blood serum of the experimental animal was analysed to determine the biochemical and metabolite changes. PB treated group demonstrated inhibition of body weight gain without showing an effect on the food intake. In serum bioassay, the PB treated group (HF/PB (100mg/kg and 500mg/kg) showed an increased in glucose and cholesterol levels compared to the Standard Diet (SD/WTR) group, a decrease in LDL level and increase in HDL level when compared with High Fat Diet (HF/WTR) group. For metabolite analysis, two separation models were made to determine the metabolite changes via group activities. The best separation of PCA serum in Model 1 and 2 was achieved in principle component 1 and principle component 2. SUS-Plot model showed that HF group was characterized by high-level of glucose, glycine and alanine. Increase in the β-hydroxybutyrate level similar with SD group animals was evident in the HF/PB(500mg/kg) group. This finding suggested that the administration of 500mg/kg PB extracts leads to increase in oxidation process in the body thus maintaining the body weight and without giving an effect on the appetite even though HF was continuously consumed by the animals until the end of the studies and also a reduction in food intake, thus maintaining their body weight although they were continuously consumed HF.
White feces syndrome (WFS) is an emerging problem for penaeid shrimp farming industries in SE Asia countries, Thailand, Malaysia, Vietnam, Indonesia, China, and in India. This occurrence of this syndrome is usually first evidenced by the appearance of white fecal strings floating on surface of the shrimp ponds. The gross signs of affected shrimp include the appearance of a whitish hindgut and loose carapace, and it is associated with reduced feeding and growth retardation. To investigate the nature of the white feces syndrome, samples of white feces and shrimp hepatopancreas tissue were collected from Penaeus vannamei in affected farms in Indonesia, and these were examined histologically. Within the white feces, we found densely packed spores of the microsporidian Enterocytozoon hepatopenaei (abbreviated as EHP) and relatively fewer numbers of rod-shaped bacteria. From WFS ponds, hepatopancreas samples form 30 individual shrimp were analyzed by histology and in situ hybridization. The results showed that all of the shrimp examined were infected with EHP accompanied by septic hepatopancreatic necrosis (SHPN). Midgut epithelial cells were also infected and this increased the number of tissue types being affected by EHP. By PCR, EHP was detected in all the samples analyzed from WFS-affected ponds, but not in those sampled from healthy shrimp ponds. To determine the modes of transmission for this parasite, we performed feeding and cohabitation bioassays, the results showed that EHP can be transmitted through per os feeding of EHP-infected hepatopancreas tissue to healthy shrimp and through cohabitation ofinfected and healthy shrimp. In addition, we found the use of Fumagillin-B, an antimicrobial agent, was ineffective in either reducing or eliminating EHP in infected shrimp.
Gracilaria species are red marine macroalgae that are found abundantly in Malaysia. Gracilaria changii from Morib, Selangor, G. nanilaensis and Gracilaria sp. from Gelang Patah, Johor were used in this study. Five compounds were successfully isolated and identified as hexadecanoic acid (1), cholest-5-en-3-ol (2), 2-hydroxymyristic acid (3), cholesteryl myristate (4) and 1-(4'-methoxyphenyl)-3-(2",4",6"-trihydroxyphenyl)-3-hydroxypropanone (5) based on spectral data analysis (IR, UV, GC-MS, 'H NMR, "C NMR, HMQC and HMBC). All compounds isolated were tested for cytotoxicity (MTT assay for HL-60 and MCF-7 cell lines), and antibacterial (disc diffusion method), antioxidant (DPPH free radical scavenging assay and xanthine oxidase inhibitory assay) and acetylcholinesterase inhibitory (AChE) activity (TLC bioautographic method). Compounds I and 3 exhibited strong cytotoxic activity against HL-60 and MCF-7 cell lines. Compound 5 showed high antioxidant activity in both the DPPH free radical scavenging and xanthine oxidase inhibition assays. Compound I showed positive activity for AChE inhibitory with a minimum inhibition dose of 0.625 tg sample. All compounds demonstrated antibacterial activity producing 8 to 14 mm inhibition zones. A positive control was applied to all bioassays and experiments were performed with three replicates. Results demonstrated that three edible red seaweeds are rich sources of bioactive compounds with potential application for pharmaceutical purposes.
The treatment of Acanthamoeba infections remains problematic, suggesting that new targets and/or chemotherapeutic agents are needed. Bioassay-guided screening of drugs that are clinically-approved for non-communicable diseases against opportunistic eukaryotic pathogens is a viable strategy. With known targets and mode of action, such drugs can advance to clinical trials at a faster pace. Recently Bortezomib (proteasome inhibitor) has been approved by FDA in the treatment of multiple myeloma. As proteasomal pathways are well known regulators of a variety of eukaryotic cellular functions, the overall aim of the present study was to study the effects of peptidic and non-peptidic proteasome inhibitors on the biology and pathogenesis of Acanthamoeba castellanii of the T4 genotype, in vitro. Zymographic assays revealed that inhibition of proteasome had detrimental effects on the extracellular proteolytic activities of A. castellanii. Proteasome inhibition affected A. castellanii growth (using amoebistatic assays), but not viability of A. castellanii. Importantly, proteasome inhibitors affected encystation as determined by trophozoite transformation into the cyst form, as well as excystation, as determined by cyst transformation into the trophozoite form. The ability of proteasome inhibitor to block Acanthamoeba differentiation is significant, as it presents a major challenge in the successful treatment of Acanthamoeba infection. As these drugs are used clinically against non-communicable diseases, the findings reported here have the potential to be tested in a clinical setting against amoebic infections.
Based on an ethnobotanical study on use for plant species against mosquito bites in the Kota Tinggi District, Johor State, Malaysia, 3 plants selected for study, Citrus aurantifolia (leaves), Citrus grandis (fruit peel), and Alpinia galanga (rhizome), were extracted using hydrodistillation to produce essential oils. These essential oils were then formulated as a lotion using a microencapsulation process and then tested for their repellent effect against Aedes aegypti. N,N-diethyl-m-toluamide (deet) was also prepared in the same formulation and tested for repellency as controls. Four commercial plant-based repellent (KAPS(®), MozAway(®), BioZ Natural(®), and Mosiquard(®)) also were incorporated in the bioassay for comparison purposes. Bioassays revealed that at 20% concentration all repellent formulations demonstrated complete protection for 2 h and >90% for 4 h post-application. The A. galanga-based formulation provided the greatest level of protection (98.91%), which extended for 4 h post-application and was not significantly different from deet at similar concentration. When compared with commercial plant-based repellents (KAPS(®), MozAway(®), and BioZ Natural(®)), the 3 lotion formulations showed significantly better protection against Ae. aegypti bites, providing >90% protection for 4 h. In conclusion, our 3 plant-based lotion formulations provided acceptable levels of protection against host-seeking Ae. aegypti and should be developed.
We evaluated the activity of methanolic extracts of Melaleuca cajuputi flowers against the filarial worm Brugia pahangi and its bacterial endosymbiont Wolbachia. Anti-Wolbachia activity was measured in worms and in Aedes albopictus Aa23 cells by PCR, electron microscopy, and other biological assays. In particular, microfilarial release, worm motility, and viability were determined. M. cajuputi flower extracts were found to significantly reduce Wolbachia endosymbionts in Aa23 cells, Wolbachia surface protein, and microfilarial release, as well as the viability and motility of adult worms. Anti-Wolbachia activity was further confirmed by observation of degraded and phagocytized Wolbachia in worms treated with the flower extracts. The data provided in vitro and in vivo evidence that M. cajuputi flower extracts inhibit Wolbachia, an activity that may be exploited as an alternative strategy to treat human lymphatic filariasis.
Lateral flow assays (LFAs) hold great promise for point-of-care testing, especially in resource-poor settings. However, the poor sensitivity of LFAs limits their widespread applications. To address this, we developed a novel device by integrating dialysis-based concentration method into LFAs. The device successfully achieved 10-fold signal enhancement in Human Immunodeficiency Virus (HIV) nucleic acid detection with a detection limit of 0.1nM and 4-fold signal enhancement in myoglobin (MYO) detection with a detection limit of 1.56ng/mL in less than 25min. This simple, low-cost and portable integrated device holds great potential for highly sensitive detection of various target analytes for medical diagnostics, food safety analysis and environmental monitoring.
Lateral flow assays (LFAs) have been extensively explored in nucleic acid testing (NAT) for medical diagnostics, food safety analysis and environmental monitoring. However, the amount of target nucleic acid in a raw sample is usually too low to be directly detected by LFAs, necessitating the process of amplification. Even though cost-effective paper-based amplification techniques have been introduced, they have always been separately performed from LFAs, hence increasing the risk of reagent loss and cross-contaminations. To date, integrating paper-based nucleic acid amplification into colorimetric LFA in a simple, portable and cost-effective manner has not been introduced. Herein, we developed an integrated LFA with the aid of a specially designed handheld battery-powered system for effective amplification and detection of targets in resource-poor settings. Interestingly, using the integrated paper-based loop-mediated isothermal amplification (LAMP)-LFA, we successfully performed highly sensitive and specific target detection, achieving a detection limit of as low as 3 × 10(3) copies of target DNA, which is comparable to the conventional tube-based LAMP-LFA in an unintegrated format. The device may serve in conjunction with a simple paper-based sample preparation to create a fully integrated paper-based sample-to-answer diagnostic device for point-of-care testing (POCT) in the near future.
In recent years, paper-based point-of-care testing (POCT) has been widely used in medical diagnostics, food safety and environmental monitoring. However, a high-cost, time-consuming and equipment-dependent sample pretreatment technique is generally required for raw sample processing, which are impractical for low-resource and disease-endemic areas. Therefore, there is an escalating demand for a cost-effective, simple and portable pretreatment technique, to be coupled with the commonly used paper-based assay (e.g. lateral flow assay) in POCT. In this review, we focus on the importance of using paper as a platform for sample pretreatment. We firstly discuss the beneficial use of paper for sample pretreatment, including sample collection and storage, separation, extraction, and concentration. We highlight the working principle and fabrication of each sample pretreatment device, the existing challenges and the future perspectives for developing paper-based sample pretreatment technique.
There is emergence of multidrug-resistant Salmonella enterica serotype typhi in pandemic proportions throughout the world, and therefore, there is a necessity to speed up the discovery of novel molecules having different modes of action and also less influenced by the resistance formation that would be used as drug for the treatment of salmonellosis particularly typhoid fever. The PhoP regulon is well studied and has now been shown to be a critical regulator of number of gene expressions which are required for intracellular survival of S. enterica and pathophysiology of disease like typhoid. The evident roles of two-component PhoP-/PhoQ-regulated products in salmonella virulence have motivated attempts to target them therapeutically. Although the discovery process of biologically active compounds for the treatment of typhoid relies on hit-finding procedure, using high-throughput screening technology alone is very expensive, as well as time consuming when performed on large scales. With the recent advancement in combinatorial chemistry and contemporary technique for compounds synthesis, there are more and more compounds available which give ample growth of diverse compound library, but the time and endeavor required to screen these unfocused massive and diverse library have been slightly reduced in the past years. Hence, there is demand to improve the high-quality hits and success rate for high-throughput screening that required focused and biased compound library toward the particular target. Therefore, we still need an advantageous and expedient method to prioritize the molecules that will be utilized for biological screens, which saves time and is also inexpensive. In this concept, in silico methods like machine learning are widely applicable technique used to build computational model for high-throughput virtual screens to prioritize molecules for advance study. Furthermore, in computational analysis, we extended our study to identify the common enriched structural entities among the biologically active compound toward finding out the privileged scaffold.
To accelerate the discovery of novel leads for the treatment of Human African Trypanosomiasis (HAT), it is necessary to have a simple, robust and cost-effective assay to identify positive hits by high throughput whole cell screening. Most of the fluorescence assay was made in black plate however in this study the HTS assay developed in 384-well format using clear plate and black plate, for comparison. The HTS assay developed is simple, sensitive, reliable and reproducible in both types of plates. Assay robustness and reproducibility were determined under the optimized conditions in 384-well plate was well tolerated in the HTS assay, including percentage of coefficient of variation (% CV) of 4.68% and 4.74% in clear and black 384-well plate, signal-to-background ratio (S/B) of 12.75 in clear 384-well plate and 12.07 in black 384-well plate, Z' factor of 0.79 and 0.82 in clear 384-well plate and black 384-well plate, respectively and final concentration of 0.30% dimethylsulfoxide (DMSO) in both types of plate. Drug sensitivity was found to be comparable to the reported anti-trypanosomal assay in 96-well format. The reproducibility and sensitivity of this assay make it compliant to automated liquid handler use in HTS applications.
Dracaena cinnabari Balf.f. is a red resin endemic to Socotra Island, Yemen. Although there have been several reports on its therapeutic properties, information on its cytotoxicity and anticancer effects is very limited. This study utilized a bioassay-guided fractionation approach to determine the cytotoxic and apoptosis-inducing effects of D. cinnabari on human oral squamous cell carcinoma (OSCC). The cytotoxic effects of D. cinnabari crude extract were observed in a panel of OSCC cell lines and were most pronounced in H400. Only fractions DCc and DCd were active on H400 cells; subfractions DCc15 and DCd16 exhibited the greatest cytotoxicity against H400 cells and D. cinnabari inhibited cells proliferation in a time-dependent manner. This was achieved primarily via apoptosis where externalization of phospholipid phosphatidylserine was observed using DAPI/Annexin V fluorescence double staining mechanism studied through mitochondrial membrane potential assay cytochrome c enzyme-linked immunosorbent and caspases activities revealed depolarization of mitochondrial membrane potential (MMP) and significant activation of caspases 9 and 3/7, concomitant with S phase arrest. Apoptotic proteins array suggested that MMP was regulated by Bcl-2 proteins family as results demonstrated an upregulation of Bax, Bad, and Bid as well as downregulation of Bcl-2. Hence, D. cinnabari has the potential to be developed as an anticancer agent.
The allelochemical 2,4-di-tert-butylphenol (2,4-DTBP) is one of the natural compounds present in medicinal plants.
This compound has been reported to possess herbicidal properties. However, its effect on weed growth parameters is
unknown for it to be utilized in weed management. Hence, the herbicidal potential of the allelochemical 2,4-DTBP on the
root and leaf tissues of the grassy weed, Leptochloa chinensis (L.) Nees and the broadleaf weed, Hedyotis verticillata
(L.) Lam was investigated. After 2,4-DTBP treatment, both bioassay species had abnormal and much shorter root hairs
compared to those of untreated plants. The roots of H. verticillata were severely damaged with the root nodes turned
brown. The phytotoxic effect of 2,4-DTBP on L. chinensis and H. verticillata became apparent at seven days and 14 days
after treatment with symptoms of lamina wilting and necrosis, respectively. These results demonstrated that 2,4-DTBP
could be used as a natural herbicide for the control of L. chinensis and H. verticillata.
Plants have been a good source of therapeutic agents for thousands of years;
an impressive number of modern drugs used for treating human diseases are derived from
natural sources. The Theobroma cacao tree, or cocoa, has recently garnered increasing
attention and become the subject of research due to its antioxidant properties, which are
related to potential anti-cancer effects. In the past few years, identifying and developing
active compounds or extracts from the cocoa bean that might exert anti-cancer effects
have become an important area of health- and biomedicine-related research. This review
provides an updated overview of T. cacao in terms of its potential anti-cancer compounds
and their extraction, in vitro bioassay, purification, and identification. This article also
discusses the advantages and disadvantages of the techniques described and reviews the
processes for future perspectives of analytical methods from the viewpoint of anti-cancer
compound discovery.
The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.
The rise in pollution cases globally is expected to increase in line with industrialization.
Monitoring activities for pollutants have been hampered by the astronomical costs of
instrumental-based approach. This has resulted in the intense research on low cost
biomonitoring systems using enzymes, organisms including microorganisms. Only positive
samples are sent for instrumental analysis; dramatically cutting the cost of instrumental
analysis. This review attempts to outline and give due recognition to several selected bioassay
systems that have been tested for their applicability using polluted water samples as a routine
first line-of-defense. This includes small aquatic organisms-based assays, enzymes especially
proteases and bacterial-based systems using respiratory dye or luminescence systems as a
method for toxicant detection.
In this work, a temporal monitoring work for heavy metals from an effluent discharge point in
the Juru Industrial Estate was carried out using the protease extracted from garlic (Allium
sativum) as the principal bioassay system. casein-Coomassie-dye binding assay method has
utilized this purpose. The periodic sampling results for one day of a location in the Juru
Industrial Estate showed temporal variation of copper concentration coinciding with garlic
protease inhibition with the highest concentrations of copper occurring between 12.00 and 16.00
hours of between 3 and 3.5 mg/L copper. The crude proteases extracted from Allium sativum
successfully detect temporal variation of copper form this location. In conclusion, this assay
method has the potential to be a rapid, sensitive, and economic inhibitive assay for the largescale
biomonitoring works for the heavy metal copper from this area.
Acetylcholinesterase (AChE) is usually used as an inhibitive assay for insecticides. A lesserknown
property of AChE is its inhibition by heavy metals. In this work, we evaluate an AChE
from brains of Clarias batrachus (catfish) exposed to wastes from aquaculture industry as an
inhibitive assay for heavy metals. We discovered that the AChE was inhibited completely by
Hg2+, Ag2+, Pb2+, Cu2+, Cd2+, Cr6+ and Zn2+ during initial screening. When tested at various
concentrations, the heavy metals exhibited exponential decay type inhibition curves. The
calculated IC50 (mg/L) for the heavy metals Ag2+, Cu2+, Hg2+, Cr6+ and Cd2+ were 0.088, 0.078,
0.071, 0.87 and 0.913, respectively. The IC50 for these heavy metals are comparable, and some
are lower than the IC50 values from the cholinesterases from previously studied fish. The assay
can be carried out in less than 30 minutes at ambient temperature.