Bioactive Phytochemical Compounds of Physalis minima L. and its Anticholinesterase and Antioxidant Activities

Physalis minima Linn. was investigated for its antioxidant and acetylcholine esterase inhibition activities. The methanolic extract of the whole plant was evaluated for radical scavenging and in vitro hydrolysis of acetylthiocholine iodide. Inhibitory concentration (IC50) of the extract exhibited IC50 values of 78.6, 46.2, 76.7 and 296 μg/mL under 2,2-diphenyl-1-picrylhydrazyl (DPPH), ABTS (2,2′-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid)), hydroxyl radical scavenging and acetylcholine esterase inhibition assays. GC-MS analysis revealed the presence of 11 compounds of which most of the compounds were reported with biological activities. The study suggests further investigations of P. minima for isolation, purification and characterization of valuable bioactive compounds related to their radical scavenging activity and for the treatment of neurodegenerative disease.

Profiling of Cholinesterase Extracted from The Brain Tissue of Diodon hystrix and Its Inhibition Reaction Towards Carbamates

Diodon hystrix, commonly known as spot-fin porcupine fish is a salt-water fish belonging to the Diodontidae family. It is widely distributed in Sabah wet market due to its commercial value. This study exploits the effectiveness of Cholinesterase (ChE) obtained from the brain tissue of D. hystrix in detecting carbamates inhibitory activities. Carbamate pesticides known to inhibit ChE and toxic towards living organisms can contaminate the water bodies. By using diethylaminoethanol (DEAE) Sepharose ion exchange chromatography, a total of 40% recovery yield of ChE was obtained with a 165.77 purification fold. Furthermore, the ChE showed a high affinity towards acetylthiocholine iodide (ATC) with an optimum activity at pH 7.45 and temperature ranging from 20 to 40℃. Among five different types of carbamates, methomyl was found to have the highest percentage of inhibition analyzed using ChE inhibitory assay, followed by carbofuran, bendiocarb, carbaryl and propoxur with >85% inhibition rate. The results concluded that ChEs extracted from the brain tissue of D. hystrix are applicable to be used as a bioindicator in detecting the presence of carbamates.

Cholinesterase from the Liver of Diodon hystrix for Detection of Metal Ions

The discharge of industrial effluents into nearby water bodies affects the inhabitants including living organisms. The presence of foreign materials such as heavy metals can be a threat to the ecosystem as they are enormously carcinogenic even though in minute concentration. Hence, an economical and time-efficient preliminary screening test is crucial to be developed for the detection of heavy metals, prior to employment of high technology instruments. In this study, cholinesterase (ChE) from Sabah porcupine fish, Diodon hystrix was purified to test for its potential as an alternative biosensor in detecting metal ions. Few enzymatic parameters including specificity of substrate, temperature and pH were applied to determine its optimal enzymatic activity. ChE enzyme was found to be more sensitive towards the presence of substrate, butyrylthiocholine iodide (BTC), in contrast to acetylthiocholine iodide (ATC) and propionylthiocholine iodide (PTC) with the effective coefficient at 7193, 3680.15 and 2965.26 Vmax/Km, respectively. Moreover, the extracted ChE enzyme showed the optimum activity at pH 9 of 0.1 M Tris-HCl and at 25°C to 30°C range of temperature. When subjected to heavy metals, ChE enzyme was significantly inhibited as the enzyme activity was reduced in the sequence of Hg > Ag > Cr > Cu > Cd > Pb ≥ Zn > As. As a conclusion, the partially purified ChE enzyme proved its sensitivity towards metal ion exposure and can be used as an alternative method in screening the level of contamination in the environment.

Functional Imaging of Neurotransmitters in Hymenolepis diminuta Treated with Senna Plant Through Light and Confocal Microscopy

Previous studies have shown the anthelmintic efficacy of Senna alata, Senna alexandrina and Senna occidentalis on the zoonotic parasite Hymenolepis diminuta through microscopic studies on morphological structure. The present study is based on the light and confocal microscopic studies to understand if Senna extracts affect neurotransmitter activity of the parasites. A standard concentration (40 mg/mL) of the three leaf extracts and one set of 0.005 mg/mL concentration of the reference drug praziquantel were tested against the parasites, keeping another set of parasites in phosphate buffer saline as a control. Histochemical studies were carried out using acetylthiocholine iodide as the substrate and acetylcholinesterase as the marker enzyme for studying the expression of the neurotransmitter of the parasite and the staining intensity was observed under a light microscope. Immunohistochemical studies were carried out using anti serotonin primary antibody and fluorescence tagged secondary antibody and observed using confocal microscopy. Intensity of the stain decreases in treated parasites compared with the control which implies loss of activity of the neurotransmitters. These observations indicated that Senna have a strong anthelmintic effect on the parasite model and thus pose as a potential anthelmintic therapy.

Carbofuran Modulating Functions of Acetylcholinesterase from Rat Brain In Vitro

Carbofuran, a potential environmental xenobiotic, has the ability to cross blood brain barrier and to adversely influence brain functions. In the present study, the impact of carbofuran on the biophysical and biochemical properties of rat brain AChE has been evaluated in vitro. This enzyme was membrane-bound which could be solubilised using Triton-X100 (0.2%, v/v), a nonionic detergent, in the extraction buffer (50 mM phosphate, pH 7.4). The enzyme was highly stable up to one month when stored at -20°C and exhibited optimum activity at pH 7.4 and 37°C. AChE displayed a direct relationship between activity and varying substrate concentrations (acetylthiocholine iodide (ATI)) by following Michaelis-Menten curve. The Km and Vmax values as computed from the Lineweaver-Burk double reciprocal plot of the data were found to be 0.07 mM and 0.066 µmole/mL/min, respectively. The enzyme exhibited IC50 value for carbofuran equal to 6.0 nM. The steady-state kinetic studies to determine mode of action of carbofuran on rat brain AChE displayed it to be noncompetitive in nature with Ki value equal to 5 nm. These experiments suggested that rat brain AChE was very sensitive to carbofuran and this enzyme might serve as a significant biomarker of carbofuran induced neurotoxicity.

The nervous systems of Tylodelphys metacercariae (Digenea: Diplostomidae) from the catfish Clarias gariepinus (Clariidae) in freshwater habitats of Tanzania

The nervous systems of three Tylodelphys metacercariae (T. mashonense, Tylodelphys spp. 1 and 2) co-occurring in the cranial cavity of the catfish, Clarias gariepinus, were examined by the activity of acetylthiocholine iodide (AcThI), with the aim of better understanding the arrangement of sensillae on the body surface and the nerve trunks and commissures, for taxonomic purposes. Enzyme cytochemistry demonstrated a comparable orthogonal arrangement in the three metacercariae: the central nervous system (CNS) consisting of a pair of cerebral ganglia, from which anterior and posterior neuronal pathways arise and inter-link by cross-connectives and commissures. However, the number of transverse nerves was significantly different in the three diplostomid metacercariae: Tylodelphys sp. 1 (30), Tylodelphys sp. 2 (21) and T. mashonense (15). The observed difference in the nervous system of the three metacercariae clearly separates them into three species. These findings suggest that consistent differences in the transverse nerves of digenean metacercariae could enable the differentiation of metacercariae to the species level in the absence of molecular techniques. This, however, might require further testing on a larger number of species of digenean metacercariae.

AcetylcholinesteraseInhibition by Biofumigant (Coumaran) from Leaves ofLantana camarain Stored Grain and Household Insect Pests

Recent studies proved that the biofumigants could be an alternative to chemical fumigants against stored grain insect pests. For this reason, it is necessary to understand the mode of action of biofumigants. In the present study the prospectus of utilisingLantana camaraas a potent fumigant insecticide is being discussed. Inhibition of acetylcholinesterase (AChE) by Coumaran, an active ingredient extracted from the plantL. camara, was studied. The biofumigant was used as an enzyme inhibitor and acetylthiocholine iodide as a substrate along with Ellman’s reagent to carry out the reactions. Thein vivoinhibition was observed in both dose dependent and time dependent in case of housefly, and the nervous tissue (ganglion) and the whole insect homogenate of stored grain insect exposed to Coumaran. The possible mode of action of Coumaran as anacetylcholinesteraseinhibitor is discussed.

Experimental Measurements for the Effect of Dilution Procedure in Blood Esterases as Animals Biomarker for Exposure to OP Compounds

Organophosphate compounds can bind to carboxylesterase, which may lower the concentration of organophosphate pesticides at the target site enzyme, cholinesterase. It is unclear from the literature whether it is the carboxylesterase affinity for the organophosphate and/or the number of carboxylesterase molecules that is the dominant factor in determining the protective potential of carboxylesterase. The fundamental dilutions and kinetic effects of esterase enzyme are still poorly understood. This study aims to confirm and extend our current knowledge about the effects of dilutions on esterases activities in the blood for birds with respect to protecting the enzyme from organophosphate inhibition. There was significantly higher esterases activities in dilution 1 : 10 in the all blood samples from quail, duck, and chick compared to other dilutions (1 : 5, 1 : 15, 1 : 20, and 1 : 25) in all cases. Furthermore, our results also pointed to the importance of estimating different dilutions effects prior to using in birds as biomarker tools of environmental exposure. Concentration-inhibition curves were determined for the inhibitor in the presence of dilutions 1 : 5, 1 : 10, plus 1 : 15 (to stimulate carboxylesterase). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Results with well-known inhibitors (malathion) were in agreement with the literature, serving to support the use of this assay. Among the thiol-esters dilution 1 : 5 was observed to have the highest specificity constant (kcat/Km), and theKmandkcatvalues were 176 μM and 16,765 s-1, respectively, for S-phenyl thioacetate ester, while detected in dilution 1 : 15 was the lowest specificity constant (kcat/Km), and theKmandkcatvalues were 943 μM and 1154 s-1, respectively, for acetylthiocholine iodide ester.

Bioactive Paper Sensor Based on the Acetylcholinesterase for the Rapid Detection of Organophosphate and Carbamate Pesticides

In many countries, people are becoming more concerned about pesticide residues which are present in or on food and feed products. For this reason, several methods have been developed to monitor the pesticide residue levels in food samples. In this study, a bioactive paper-based sensor was developed for detection of acetylcholinesterase (AChE) inhibitors including organophosphate and carbamate pesticides. Based on the Ellman colorimetric assay, the assay strip is composed of a paper support (1×10 cm), onto which a biopolymer chitosan gel immobilized in crosslinking by glutaraldehyde with AChE and 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB) and uses acetylthiocholine iodide (ATChI) as an outside reagent. The assay protocol involves introducing the sample to sensing zone via dipping of a pesticide-containing solution. Following an incubation period, the paper is placed into ATChI solution to initiate enzyme catalyzed hydrolysis of the substrate, causing a yellow color change. The absence or decrease of the yellow color indicates the levels of the AChE inhibitors. The biosensor is able to detect organophosphate and carbamate pesticides with good detection limits (methomyl=6.16×10-4 mM andprofenofos=0.27 mM) and rapid response times (~5 min). The results show that the paper-based biosensor is rapid, sensitive, inexpensive, portable, disposable, and easy-to-use.