Since ILG has been reported to reduce dopaminergic neurodegeneration and psychostimulant-induced toxicity (both of which are related to dopamine and vasopressin receptors), we investigated the binding affinity and modulatory functions of ILG on dopamine and vasopressin receptors. ILG was explored as an antagonist of the D1 receptor and an agonist of the D3 and V1A receptors with good potency. An in silico docking investigation revealed that ILG can interact with active site residues at target receptors with low binding energies. These activities of ILG on hMAO and brain receptors suggest the potential role of the compound to ameliorate dopaminergic deficits, depression, anxiety, and associated symptoms in Parkinson’s disease and other neuronal disorders.Isoliquiritigenin  (ILG) is a major constituent of the Glycyrrhizae Rhizoma that has significant neuroprotective functions. In the present study, we re-examined the potential of ILG to inhibit human monoamine oxidase (hMAO) in vitro and established its mechanism of inhibition through a kinetics study and molecular docking examination. ILG showed competitive inhibition of hMAO-A and mixed inhibition of hMAO-B with IC Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the common neurodegenerative disorders; each has a prevalence of approximately 1% of the elderly population in their late sixties and even more in the latter decades of life2. PD is a nervous system disorder characterized by progressive degeneration of nigrostriatal dopaminergic neurons in the midbrain, resulting in depletion of striatal dopamine (DA). Motor symptoms  are the hallmarks of PD and often are accompanied by non-motor features, like cognitive dysfunction, depression, anxiety, and other socio-behavioral anomalies. Multiple mechanisms that provoke neurodegeneration have been proposed, such as complex molecular changes involving DA and non-dopamine neurons (such as cholinergic and gamma-aminobutyric acid-B [GABA]ergic neurons) and non-neuronal cells (such as microglia and astrocytes), perturbed cell functions like mitochondrial dysfunction resulting from excessive reactive oxygen species (ROS) or either underexpression or mutation of protective genes like peroxisome proliferator-activated receptor-gamma activator-1α (PGC-1α) and parkin-associated proteins . In addition, α-synuclein aggregation is a pathological marker of PD. Overall, the pathogenesis of PD represents a complex multifactorial process involving neurodegeneration and progression of the disease4.Neurodegenerative diseases (NDDs) include a heterogeneous set of disorders characterized by progressive and irreversible damage to the neurons of the central nervous system (CNS) that can lead to functional and mental impairmentsl-Dopa) often is combined with a DA receptor agonist, a monoamine oxidase-B (MAO-B) inhibitor, or a catechol-O-methyltransferase inhibitor to allay motor symptoms in advanced PD. Such combination nevertheless holds drawbacks, such as polypharmacology-related adverse drug reactions due to varying pharmacokinetic and pharmacodynamics profiles of each drug and medication non-adherence and noncompliance. Thus, another multi-target strategy in which a single chemical entity can influence more than one target has piqued the interest of medicinal chemists. This approach can avoid adverse effects associated with combination drugs and attain a more predictable pharmacokinetic profile of a single drug compared to the multiple drugs administered in combination therapy5.For multifactorial diseases like PD that have intertwined pathophysiological routes, the “one gene, one target, one drug” paradigm might be inadequate to achieve the required therapeutic effect. Therefore, multi-target treatment strategies like combination therapy  that combines two or more drugs with an independent mechanism of action to obtain either an added or a synergistic effect are common. For instance, levodopa  possess multiple pharmacological activities  owing to the constitution of a variety of bioactive constituents including chalcones , isoflavonoids (licoricidin and glabridin), flavanones , the prenyl flavanoid glycycoumarin, the triterpene glycyrrhetinic acid, and the saponin glycyrrhizin7. Of these, isoliquiritigenin (ILG)  represents one of the most pharmacologically important components of the Glycyrrhiza root and its chemical structure is presented in Fig. 10. Recently, multiple studies have explored the promising effects of ILG as a neuroprotective and neurorescueing compound through inhibition of intracellular ROS generation; antioxidative action; attenuation of synaptic dysfunction, neuronal damage, and neuroinflammation, confirming its usefulness in neurodegenerative diseases14.Licorice is an ancient medicinal herb constituting three main species: 15. Thus, the inhibitors of MAO can be used as both prophylactic and therapeutic agents in neurodegenerative disorders, including AD, PD, anxiety, depression, and schizophrenia, where a loss of monoamines is observed19. Earlier studies have shown that ILG can inhibit MAOs, however, its inhibitory potential varies according to animal models. ILG displayed a weak inhibition of MAO from bovine serum (50% inhibition of MAO activity at concentrations > 200 µM)20. Whereas, ILG isolated from Sinofranchetia chinensis demonstrated significant inhibition of rat brain MAO-A and MAO-B (rMAO-A/B), with IC50 values of 13.9 and 47.2 µM, respectively21. ILG inhibited human MAO-A (hMAO-A) and human MAO-B (hMAO-B), with IC50 values 41.5 and 21.8 µM, respectively22.Monoamine oxidases (MAOs) are the principal flavoproteins responsible for the catalytic breakdown of monoamine neurotransmitters, and two isoforms of MAO (MAO-A and MAO-B) are present in human tissues. Serotonin and norepinephrine are selectively metabolized by MAO-A, whereas phenylethylamine and benzylamine are preferentially degraded by MAO-B. DA, tyramine, and tryptamine are deaminated by both forms of MAOs. on the hMAO inhibitory activities of ILG derivatives found that ILG is selective toward hMAO-B, in contrast to higher selectivity toward rMAO-A. The synthesized compound C8 showed higher potency and selectivity for hMAO-B than ILG; the enzymatic activity of C8 against hMAO-B was about 16 times greater than that of ILG. However, molecular docking showed greater proximity of ILG toward the catalytic site active residues of hMAO-B than of C824. To examine the incongruent results between the enzymatic activity and the in silico docking study and to investigate the reproducibility of the inhibitory action of ILG on recombinant hMAOs, we re-evaluated the functions of ILG on hMAO-A and hMAO-B using a homogenous luminescent assay, enzyme kinetics, and molecular simulation studies. The present work includes a detailed study of inhibition kinetics and an in silico docking study focusing on the ILG-hMAO (A/B) complex, which has not been reported.A study by Zhuo et alB) receptor25 and also, inhibit N-methyl-d-aspartate receptor-induced Ca2+ influx26. Pretreatment with ILG controlled the hyperlocomotion caused by methamphetamine (METH) treatment in mice but did not cause a significant change in the monoamine levels in tissues of the cerebral cortex, striatum, nucleus acumen, thalamus, or hypothalamus27. METH is a psychoactive agent that increases the extracellular DA level either by suppressing DA reuptake or internalization of dopamine transporter (DAT) from the plasma membrane or stimulation of DA efflux, and results in rewarding and addictive effect28. Repeated administration of METH was demonstrated to affect the nigrostriatal DA pathway causing the neurodegeneration of dopaminergic terminals and subsequent reduction in tyrosine hydroxylase, DAT, and striatal DA levels, predisposing the patient to PD. Dopamine receptors (DARs) mediate various cognitive and behavioral functions associated with DA, and DA D1 type receptor (D1R and D5R) antagonism was found to prevent amphetamine-induced striatal neurodegeneration30. In addition, Everett et al. demonstrated that V1AR plays a substantial role to mediate the inhibitory action of oxytocin on METH-primed reinstatement and drug-seeking behaviors31. Thus, our present study aimed to assess the plausible role of ILG on G-protein coupled receptors (GPCRs), such as DARs , and the vasopressin receptor (V1AR) via radioligand binding and functional GPCR assays.ILG was found to suppress cocaine-induced DA release in the rat brain by modulating the gamma-aminobutyric acid-B ; as Kic < Kiu, the compound has a greater affinity to inhibit unbound enzymes compared to substrate-bound enzymes.ILG exhibited a strong inhibitory effect against hMAO-A and hMAO-B, as indicated by the low ICes Table . ILG inhThe overall docking results including the binding energy and the interacting residues of hMAO-A and hMAO-B when ILG binds to these enzymes are shown in Table ILG interacted with the catalytic site residues of hMAO-A with a binding energy of − 7.44 kcal/mol, which was lower than that required for the standard ligand harmine . The docking pose revealed that the 2′,4′-dihydroxyphenyl moiety of ILG occupied the central catalytic site of the hMAO-A and interacted with Tyr407 via a π-π T-shaped hydrophobic bond, and with Tyr444 and flavin adenine dinucleotide (FAD) via H-bonds. A carbonyl group at C1 was aligned toward FAD, which enabled its interaction with FAD through an H-bond. The aromatic ring of the 4″-hydrophenyl moiety engaged in hydrophobic interactions with active site residues Ile335, Leu337, and Phe208 through π-σ and π–π stack bonds. A H-bond between 4″-OH and Thr336 was also formed in the ILG‒hMAO-A complex.23. Molecular analysis revealed that ILG could bind with the residues of the substrate-binding cavity and the hydrophobic cavity between the substrate cavity and entrance cavity of the enzyme with a binding energy of − 8.69 kcal/mol. The 4″-hydroxyphenyl moiety of the compound interacted with Tyr398 and FAD, while another aromatic ring  was aligned to interact with gating residues Leu171 and Ile199. Pi-sulfur and H-bond interactions were formed with Cys172, a residue in the hydrophobic cavity, via 2′,4′-dihydroxyphenyl moiety and 1-CO group, respectively. In addition to catalytic interactions, ILG formed bonds with allosteric site residues of hMAO-B, with − 8.03 kcal/mol of binding energy -D1R, human embryonic kidney (HEK)-D2LR, CHO-D3R, and CHO-D4R cell membranes. The V1AR binding property was evaluated by the agonist radioligand binding assay using [3H] arginine vasopressin (AVP) on the V1AR-transfected CHO cells and validated using AVP as a reference. Screening the binding property of ILG at 100 μM revealed significant inhibition of control-specific binding (> 50%) for D1R, D3R, and V1AR and moderate inhibition of D4R with 44.8% inhibition of control-specific binding (Table 1R membrane (96.0%), whereas no binding affinity was seen on the HEK-D2LR membrane.The binding affinity of ILG to the human dopaminergic D1R, D3R, and V1AR receptors. Since binding assays are limited to determining the affinity and selectivity of ligands to receptors, we conducted cellular functional assays to reveal agonist or antagonist behavior of ILG on D1R, D3R, and V1AR. Functional GPCR assays showed that ILG is a D1R antagonist and a D3R and V1AR agonist. As shown in Table 1R control agonist response by 91.9 ± 3.1% and stimulated D3R and V1AR by 92.7 ± 0.5% and 73.3 ± 4.0% of the control agonist response, respectively. Figure 1R and the percent of the control agonist response on D3R and V1AR. The concentration required to produce 50% of agonist response (EC50) or antagonist response (IC50) of ILG along with those of the reference drugs on the tested receptors are presented in Table 1R antagonist, ILG had an IC50 value of 68.9 ± 0.3 µM, whereas, for D3R and V1AR stimulation, the EC50 values were 62.3 ± 0.2 and 78.6 ± 2.7 µM, respectively.The preliminary in vitro binding screening using 100 μM ILG indicated a high binding affinity for the D1R, D3R, and V1AR, computational docking analyses were carried out using AutoDock 4.2. Figures 1R, D3R, and V1AR, respectively. Overall ligand-receptor interactions, including H-bonding, electrostatic and hydrophobic interactions, and binding scores, obtained from the docking of the receptors with the ILG and the reference ligands are presented in Table 1R (hD1R) and interacted with residues Asn292, Asp103, and Ile154 through H-bonds to three hydroxyl groups at C2′, C4′, and C4″ of ILG. Two phenyl rings of the chalcone moiety anchored with conserved residues Ile104, Phe288, and Ser198 of transmembranes (TM) 3, 6, and 5, respectively, via strong hydrophobic connections.To examine the possible interactions of ILG at the binding sites of D3R revealed that ILG can bind to the orthosteric binding site (OBS) of D3R with a lower binding energy  than that required for the reference agonist DA . Polar interaction of 4′-OH was found with Asp110 of TM3, which was similar to the case of DA but without the formation of a salt bridge. Major interactions were observed with the amino acid residues of TMs 3 and 5, such as Val107  and Val189 . An electrostatic π-cation linkage with the key OBS residue His348 was formed with the 4″-hydroxyphenyl moiety. Moreover, ILG extends toward the second extracellular loop (ECL2) and binds to Cys181 via a H-bond and to Ile183 via a non-polar bond.Molecular docking of ILG with D1AR with a binding energy of − 7.03 kcal/mol, which is lower than that required for AVP binding . In silico docking displayed five hydrophilic connections of ILG with the residues of V1AR that were assisted by the 2′,4′,4″-hydroxy functional groups and a carbonyl group at C1. An electrostatic interaction of the 4″-hydroxyphenyl moiety with a non-conserved hydrophilic residue (Asp112 of TM2) was observed. Hydrophobic interactions of the 2′,4′-dihydroxy phenyl ring of ILG occurred with the Val127 and Lys128 residues of TM3. Of all molecular interactions, Glu54, Asp112, and Lys128 were the common binding residues compared with the reference agonist AVP.Similarly, ILG was predicted to be buried within helices 1, 2, and 3 of VPharmacokinetic parameters and toxicity of ILG were predicted using SwissADMET and pkCSM applications. SwissADMET showed drug-likeness and lead-likeness of ILG with a lipophilicity of 2.37 (log Po/w) and a solubility of − 3.06 (soluble). A pkCSM application indicated high intestinal absorption (HIA) of ILG (> 90%) along with its probability to cross the blood brain barrier (BBB) and reach the CNS. It also predicted ILG as a safe drug-like molecule with no toxicity to hepatocytes or upon Ames testing  values with respect to the IC50 values for each enzyme  to have very potent action against hMAO isoenzymes compared to its activity against rMAO isoenzymes34. In addition, screening of coumarin and 5H-indenopyridazin-5one derivatives for inhibition of MAOs of human and rat showed greater effectiveness of compounds against hMAO-B than against rMAO-B and no correlation between human and rat pIC50 values35. These findings support that results drawn from experimentation in one animal model cannot consistently be extrapolated to humans.Although reports on hMAO inhibition by ILG are available, a detailed study on the mechanism of hMAO inhibition is lacking. Thus, our study incorporated an enzyme kinetics study of ILG for inhibition of hMAO isoenzymes and demonstrated concordant inhibition constant  with a higher affinity for hMAO-B 36. Other natural chalcones, such as 4-hydroxyderricin, 2,2′-dihydroxy-4′,6′-dimethoxychalcone, and broussochalcone, also have notable hMAO inhibition potential36.Our docking study showed good proximity of ILG toward the isoalloxazine of the FAD cofactor in hMAO-A and hMAO-B. ILG bonded with key residues of the hMAO isoenzymes through multiple polar and non-polar interactions that resulted in strong inhibition. In hMAO-A, the 2′,4′-dihydroxyphenyl ring was engaged in interaction with the major active site residues FAD, Tyr444, and Tyr407, while in the case of hMAO-B, the 4-hydroxyphenyl moiety was responsible for forming strong interactions with FAD and a catalytic residue Tyr398. The carbonyl oxygen of ILG assisted in the formation of a stable MAO-ILG adduct through H-bonding with important active site factors (FAD in hMAO-A and Cys172 in hMAO-B). LG, a structurally interrelated flavanone form of ILG, was reported to have strong inhibition of hMAO-A and hMAO-B, with IC37, 6–OHDA38, and glutamate-induced neurotoxicity39 and alleviate cognitive impairment14, anxiety, and locomotor sensitization40. Since the modulation of DA and vasopressin receptors has been found to affect cognition, anxiety, and neuronal survival, we evaluated the binding affinity of ILG to the D1R, D2R, D3R, D4R, and V1AR receptors to determine whether ILG can target these receptors and identified significant binding to D1R, D3R, and V1AR. The functional GPCR assay showed ILG to have D1R antagonist and D3R and V1AR agonist properties. A ligand-D1R interaction study revealed that ILG can bind with the active site residues Asp103, Ile104, and Phe288 similar to the reference antagonist SCH23390. It has been reported that interactions with Asp103, Asn292, and Ser198 residues are vital for strong binding affinity of the ligand to the D1R41. The molecular docking pose of D3R with ILG indicates that ILG extends between helices 3, 5, and 6 to the ECL2. Some ECL2 residues (182–185) form a part of the ligandbinding pocket of D3R, and interactions with ECL2 residues have been acknowledged for their selectivity of ligands between D2 and D3 receptors42. Thus, the binding of ILG with Cys181 and Ile183 of ELC2 might be responsible for the higher affinity of ILG for D3R compared to D2R. Cotte et al. reported that aromatic residues in V1AR play an insignificant role in the agonist’s binding affinity; however, the conserved hydrophilic residues Glu108, Lys128, and Gln185 contribute significantly to the V1AR agonist AVP binding43. The H-bonding with Gln108 and non-polar linkage with Lys128, along with other known agonist binding residues 44, were detected by computational docking of ILG with a 3D model of human V1AR.In vitro and in vivo studies have shown that ILG can protect dopaminergic neurons from METHl-Dopa is the gold standard therapy; however, prolonged use of l-Dopa causes dyskinesia and dystonia. DAR agonists stimulate the postsynaptic DARs and enhance the function of the DA system. Thus, they are considered the first choice in de nova patients to delay the onset of l-Dopa therapy45. Another pharmacological principle used to alleviate DA deficiency in PD is the use of reversible and irreversible hMAO-B inhibitors, such as selegiline, rasagiline, and safinamide. Clinical trials have shown improvement of motor symptoms when safinamide was used as an adjunct to l-Dopa or single DA receptor agonist therapy46. MAO-B as well as MAO-A inhibitors have neuroprotective functions since they suppress the oxidation of monoamines and prevent the resulting generation of ROS and neurodegeneration in NDDs, such as PD and AD. MAO-A inhibitors such as iproniazid and tranylcypromine are clinically indicated for depression. Moreover, MAO-A inhibition shows potential protective roles in cancer and myocardial damage47.For treatment of PD, l-Dopa concentration in the plasma and the pulsatile stimulation of DARs, DAR agonists can be used as an add-on therapy to l-Dopa48. The D2/D3 receptor agonist pramipexole was found to improve depression in PD, anhedonia, and motor deficits49. D1R activation has shown improvement in hippocampal neurogenesis and anxiolytic and antidepressant-like effects in a rat model of PD, whereas the D1R antagonist SCH23390 was found to inhibit stimulant-induced DAR supersensitivity50. Addictive psychostimulants like cocaine, amphetamines, and METH increase D1R activation, leading to the amplification of c-fos and fosB expression in the striatum. These elevations in c-fos and fosB along with the METH-induced increases in c-Jun, JunB, JunD, and Fra2 expression were all inhibited by pretreatment with the D1R antagonist SCH2339051. Although the expression of D1Rs is reduced in PD, increased stimulation of D1R signaling pathways has been linked to l-Dopa-induced dyskinesia52. The activation of D1Rs by METH is known to cause neurotoxicity of nigrostriatal dopaminergic neurons, and the deletion of D1Rs or D1R antagonism considerably protected against METH-induced neurotoxic effects53. Thus, the neuroprotective effects of ILG, which had previously been shown to impede METH-induced neurotoxicity37 and hyperlocomotion without alterations in monoamine levels27, can be attributed to its D1R antagonist behavior and MAO-inhibition potential.To treat the motor and non-motor complications of PD that can arise from fluctuations in 3R agonists (such as 7-OH-DPAT) can restore nigrostriatal integrity by inducing neurogenesis and therefore improve locomotor function in PD models54. Piribedil, another D3R agonist, was found to be effective in enhancing learning and memory in an animal model of cerebral ischemia-reperfusion55. Vasopressin V1AR agonist activity has been associated with cognitive function and social recognition, while V1AR antagonists have an anxiolytic effect. Studies conducted on V1AR knockout mice have identified abnormalities in social interaction and recognition on social behavior tests and reduced anxiety levels in elevated plus-maze and marble-burying behavior tests57. Therefore, the modulatory effects of ILG on V1AR might be related to reduced anxiety-like behavior, which was observed following ILG treatment in nicotine-withdrawn mice40.Former investigations in neuroscience have reported that D58 and induces Nfr2-dependent detoxification genes59. Licorice is reported to be used as an adjuvant agent to enhance the efficacy of other drugs, however, herb-drug interactions have also been expected with its prolonged use. ILG, as one of the phytoconstituents, has been found to inhibit cytochrome P450 enzymes such as CYP1A2, CYP2C19, CYP2C9, and CYP3A4. These enzymes are responsible for major drug metabolism, and inactivation of these enzymes can reduce drug metabolism, thus, leading to increased plasma concentration and the risks of side effects60. Treatment with higher doses (36 and 100 µM) of ILG inhibited follicular growth and steroidogenesis owing to the perturbation in the expression of key steroidogenesis regulators such as CYP17A1, CYP19A1, and HSD17B161.The computational prediction of pharmacokinetic parameters such as drug-likeness, solubility, absorption, and CNS permeability for ILG shows favorable characteristics. In silico prediction also reveals no toxicity of ILG on AMES test and hepatotoxicity assessment. Experimentally, ILG has been found to be hepatoprotective1, D3, and V1A receptors. Probing the modulatory activity of ILG on DA (D1 and D3) and vasopressin (V1A) receptors revealed that ILG has an antagonist effect on the D1 receptor and agonist effects on the D3 and V1A receptors. The multi-target nature of ILG together with the prediction of suitable pharmacokinetics and the toxicity profile renders this compound as a potential flavonoid for the management of PD and its related neurological symptoms. Future experimental studies on ILG’s pharmacological and toxicological properties and adverse effects will be important to confirm its therapeutic efficacy and benefits.Overall, this study reinvestigated the hMAO-A and hMAO-B inhibitory activity of ILG and established the enzyme inhibition mode through kinetic assays and computational docking. Our study recognized that ILG is a potent hMAO-inhibitor showing competitive inhibition against hMAO-A and a mixed mode of inhibition against hMAO-B. ILG was observed to have a significant binding affinity for the Dl-deprenyl⋅HCl  were used. For the GPCR functional assay, reference drugs of DA, serotonin, AVP, clozapine, butaclamol, SCH23390, (S)-WAY-100635 and -AVP as well as the test compound, ILQ, were purchased from Sigma-Aldrich . Transfected CHO and human embryonic kidney (HEK-293) cell lines were generated by Eurofins Discovery . The cell culture media of Roswell Park Memorial Institute (RPMI-1641), Dulbecco's modified Eagle medium (DMEM) buffer, Hank’s balanced salt solution (HBSS) buffer, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer were procured from ThermoFisher Scientific (USA).For the recombinant hMAO inhibition assay, a MAO-A/B assay kit , recombinant hMAO isoenzymes, and the standards 62. Briefly, we added 12.5 µl of the test compound or l-deprenyl to an aliquot of 12.5 µl of beetle luciferin derivative substrate  in each well of the plate. A 25-µl enzyme solution was added to the test samples to initiate the reaction. After an hour of incubation of the mixture at 25 °C, 50 µl reconstituted luciferin detection reagent was added to every well to stop the hMAO reaction, and an additional 20 min of incubation at 25 °C was performed. Then, the luminescence reading was taken on a FilterMax F5 Multi-Mode microplate reader .Experimental conditions and procedures for this experiment were as describedKi) for each enzyme inhibition were obtained from secondary plots analyzed using SigmaPlot 12.0 TM software .The enzyme inhibition kinetics were analyzed using varying concentrations of hMAO substrate (40–160 μM for hMAO-A and 4–16 μM for hMAO-B) and ILG (0–1 μM), as shown in Fig. 1R, D3R, and D4R binding assays, membrane homogenates (8–80 µg) of the respective receptor-expressed CHO cells were suspended in a buffer containing 50 mM Tris–HCl, 5 mM KCl, 5 mM MgCl2, 1.5 mM CaCl2/120 mM NaCl, and 5 mM EDTA and incubated for 60 min at 22 °C with appropriate radioligand [3H]SCH23390 or [3H]methylspiperone either in the presence or absence of the test compound. The human D2LR binding experiments were run on membrane preparations prepared from HEK cells. The plasma membrane homogenates (40 µg) of D2LR-expressing HEK cells were incubated with 0.3 nM [3H] methylspiperone for 60 min at 22 °C after being suspended in a binding buffer containing 50 mM Tris–HCl, 5 mM MgCl2, 1 mM EDTA, 1 UI/mL ADA, 1 µg/ml leupeptin, 1 µM pepstatin, and 10 µg/mL trypsin inhibitor. Human V1AR binding was assessed using membrane homogenates of transfected CHO cells suspended in a buffer containing 5 mM Tris–HCl, 5 mM MgCl2, and 0.1% BSA and incubated with [3H]AVP at 22 °C for 60 min. 1 µM SCH23390 (for D1R), 10 µM (+)-butaclamol , and 1 µM AVP (for V1AR) were used to define non-specific binding.The binding assays were carried out using validated methods and standard operating procedures used by Eurofins Cerep . For the human DFollowing incubation, the binding reaction within the samples was terminated by vacuum filtration through 0.3% polyethyleneimine-treated glass fiber filters . The filters were washed several times with an ice-cold wash buffer (50 mM Tris–HCl) using a 96-sample cell harvester  and dried. Scintillation cocktail  was added to the dried filters, and their radioactivity was determined using a scintillation counter . The radioligand binding results were expressed as the percent inhibition of control specific binding given by the following equation: 100 – [(measured specific binding/control specific binding) × 100].50 values were determined. The binding affinity of ILG to the receptors was screened at 100 μM.To verify the results, the standard compounds were tested at different concentrations in each of the binding assays to obtain competition curves from which their IC1R, D3R, and V1AR). The in-house assay protocol for the assay was as described in our earlier report63. The results of the functional assay were based on the measurement of effects on the cAMP level and calcium ion mobilization.Cellular and nuclear receptor functional assays were performed at Eurofins Cerep using human recombinant CHO cells transfected with the GPCR genes of interest (D1 and D3 receptors were suspended in a medium containing an HBSS buffer that had been supplemented with 20 mM HEPES buffer and 500 µM IBMX. This cell suspension was distributed into assay plates at a density of 5 × 103 cells/well and incubated at 25 °C (for the D1R transfected cell suspension) or 37 °C (for the D3R transfected suspension) for 10–30 min both with and without ILG or standard. Then, the D2-labeled cAMP conjugate was dispensed into the cell plate followed by the addition of europium cryptate-labeled anti-cAMP antibody. A lysis buffer was added to each well, and the resulting mixture was incubated for 1 h at 25 °C. The homogeneous time-resolved fluorescence (HTRF) reading was recorded using a PerkinElmer Envision microplate reader  at an excitation intensity of 337 nm and emission intensities of 620 and 665 nm. The cAMP level was calculated as the ratio of the signal measured at 665 nm to that measured at 620 nm. The final results were illustrated as the percentage of the control agonist response and as the percentage inhibition of the control agonist response.Stably transfected CHO cells containing the cDNA of human D1AR. Transfected CHO cells were suspended in an HBSS/20-mM HEPES buffer and distributed into microplate wells at a density of 1 × 105 cells/well. A fluorescent probe  was mixed with probenecid in HBSS/20 M HEPES (pH: 7.4) discretely and applied to each well, and left to equilibrate with the cells at 37 °C for 60 min. Thereafter, the assay plate was kept in a microplate reader , and ILG, the reference agonist, or HBSS buffer (control) was added to the plate; finally, the fluorescence was measured. The agonist effect was determined as the percentage of the control response to 1 µM AVP, while the antagonist behavior was calculated as the percentage inhibition of the control response to 10 nm AVP.Calcium ion influx was determined fluorimetrically to establish the functional effect of ILG on V1R and hV1AR were retrieved from the UniProt database with respective IDs of P21728 (DRD1_HUMAN) and P37288 (V1AR_HUMAN). Due to the high sequence similarity in the binding site and the overall structure between DA D1R and β2 adrenergic receptor (β2R), the model for this experiment was derived from the template of the β2R crystal structure obtained from Protein Data Bank (PDB) ID 2RH1 using the Swiss-Model server64. In the case of hV1AR, a μ-opioid receptor (PDB: 4DKL) was selected for model building66. The model was constructed based on the target (hV1aR)-template (4DKL) alignment using the Swiss-Model server67. The ModRefiner server was used to refine the model68.The sequences for hD69. X-ray crystallographic structures of hMAO-A, hMAO-B, and hD3R were attained from the RCSB PDB with IDs 2BXR, 2V60, and 3PBL, respectively. The three-dimensional chemical structure of ILG and the reference compounds were derived from the PubChem Compound database (NCBI). Discovery Studio  was used for protein preparation. The AutoDockTool was utilized for adding necessary parameters like Gasteiger charges and rotatable bonds to perform docking simulations. Grid maps were generated by the AutoGrid program. The protocols for both rigid and flexible ligand docking consisted of 10 independent generic algorithms. The docking pose with the lowest binding score was chosen, and the results were visualized using Discovery Studio. Internal motion of the receptor during docking performance was not considered.The molecular docking study was carried out using AutoDock 4.2http://biosig.unimelb.edu.au/pkcsm/prediction)70, whereas the lipophilicity, solubility drug-likeliness, and lead-likeliness of ILG were predicted by SwissADME (http://www.swissadme.ch)71.HIA, BBB, and CNS permeability, and toxicity profile were predicted using a web-based pkCSM application (No animal model was used for the study. The study was based on in vitro enzyme and cellular experiments, and in silico study.


Dear Editor,3 Oligomerized MLKL translocates to cellular membranes, where it disrupts membrane integrity causing necroptotic cell death.3Necroptosis, a form of caspase-independent cell death, is tightly regulated to maintain tissue homeostasis. The execution of necroptosis depends on receptor interacting protein kinase 3 (RIP3)-activated mixed lineage kinase domain-like protein (MLKL). MLKL is phosphorylated by RIP3, which releases MLKL autoinhibition and drives its self-oligomerization.4 Previous mutagenesis studies implied the existence of potential inhibitory phosphorylation on MLKL.5 However, the physiological relevance of inhibitory phosphorylation on MLKL is largely unknown. Taking into account the commonly observed phenomenon that in vitro necroptosis induction cannot inflict 100% cell death in any cell lines, we harvested the necroptosis surviving cells, which were assumed to enrich the potential negative regulatory signals, and performed SILAC experiment to quantitatively characterize the phosphorylation modifications on MLKL  ,1 we also found that the cell death was blocked but RIP3-mediated MLKL phosphorylation was not affected by the S83E mutation over time (Supplementary information, Fig. The Ser83 located in the N-terminal helix bundle domain of MLKL Fig. . Previou9 It is worth noting that the D139V mutation acts as a dominant active mutation for MLKL, while the S82/83 G mutation is not (Fig. Necroptosis execution depends on membrane integration of RIP3-activated MLKL. Using Triton-114 phase separation assay, we found that the phospho-mimic mutant MLKL-S83E could not integrate into the detergent phase as WT or S83G mutant MLKL Fig. . The dirnot Fig. . InteresSupplementary information, Data and Figures


A chip-based electrochemical biosensor is developed herein for the detection of organophosphate (OP) in food materials. The principle of the sensing platform is based on the inhibition of dimethoate (DMT), a typical OP that specifically inhibits acetylcholinesterase (AChE) activity. Carbon nanotube-modified gold electrodes functionalized with polydiallyldimethylammonium chloride (PDDA) and oxidized nanocellulose (NC) were investigated for the sensing of OP, yielding high sensitivity. Compared with noncovalent adsorption and deposition in bovine serum albumin, bioconjugation with lysine side chain activation allowed the enzyme to be stable over three weeks at room temperature. The total amount of AChE was quantified, whose activity inhibition was highly linear with respect to DMT concentration. Increased incubation times and/or DMT concentration decreased current flow. The composite electrode showed a sensitivity 4.8-times higher than that of the bare gold electrode. The biosensor was challenged with organophosphate-spiked food samples and showed a limit of detection (LOD) of DMT at 4.1 nM, with a limit of quantification (LOQ) at 12.6 nM, in the linear range of 10 nM to 1000 nM. Such performance infers significant potential for the use of this system in the detection of organophosphates in real samples. Organophosphorus compounds (OPs), including pesticides and chemical nerve agents, are harmful to human health. In agriculture, the uncontrolled and excessive use of OP can contaminate harvest crops and processed foods, and organophosphates are indeed widely used in crops for both human consumption and as animal feed. OPs, neuronal acetylcholinesterase (AChE) inhibitors, have been found to contaminate raw milk and infant formulas ,2. The p4 s−1 makes it one of the most efficient oxidative/redox enzymes, allowing the catalysis of released choline in a submillisecond time frame O) , respectThrough amperometry measurements, catalytic activity and inhibition patterns (reversibility and irreversibility) of ATC and DMT were measured, respectively. DMT inhibits AChE catalytic activity, where the amount of enzymatic thiocholine product is reduced during the inhibition process. On both bare Au and composited electrodes, resulting enzymatic thiocholine is well known to adsorb through Au-S covalent bonding. This, in turn, promotes the electrolysis process of enzymatic thiocholine on electrode surfaces ,55. Cyclpc + 0.24 V and Epa +0.31, appeared regularly in the scans. The reversible reduction peak corresponds to the reduction of the thiol group to form dithiobischoline. This reaction pathway is consistent with those described elsewhere for acetylthiocholine bioconversion  (M at 0.4 (μM) was determined at 0 μM of DMT. As shown in max) value is constant at 2.5 for all DMT concentrations, but the apparent value of KM (KM/Vmax) increases with increasing DMT concentration. This result demonstrates that DMT inhibits AChE under competitive inhibition. A previous study reported DMT molecules bind to the active site  of AChE by forming a covalent bond with the serine residue at the active site  b. A valuive site . DMT molive site . HoweverWe present a sensitive fluidics-based amperometric biosensor for detecting DMT, a typical OP, working on composited electrodes. The formation of conductive nanostructures on nanocellulose matrices provides a promising microenvironment for retaining AChE activity. The electrocatalytic promotion of MWCNT contributes to enzymatic thiocholine electrolysis on the electrode surface. The composited electrode integrated into a fluidic chip was used to detect DMT as low as 4.1 nM DMT. The developed biosensor shows good sensitivity, precision, and reproducibility  for DMT-spiking samples. However, this biosensor design features significant advantages in the simplicity of the fabrication and provision of an effective immobilization platform for enzymes. This platform could be used to study enzyme assays of inhibitors for a wide range of applications.


AP) or posterior-anterior (PA) current direction over the primary motor cortex appear to activate distinct excitatory inputs to corticospinal neurons. In contrast, very few reports have examined whether the inhibitory neurons responsible for short-interval intracortical inhibition (SICI) are sensitive to TMS current direction.Pulses of transcranial magnetic stimulation (TMS) with a predominantly anterior-posterior (AP and PA conditioning stimuli (CSPA and CSAP) activate different inhibitory pathways. SICI was always assessed using a PA-oriented test stimulus (TSPA).To investigate whether SICI evaluated with PA and CSAP were applied at interstimulus intervals (ISI) of 1–5 ms before a TSPA, and at a range of different intensities. Using a triple stimulation design, we then tested whether SICI at ISI of 3 ms using opposite directions of CS (SICICSPA3 and SICICSAP3) interacted differently with three other forms of inhibition, including SICI at ISI of 2 ms (SICICSPA2), cerebellum-motor cortex inhibition (CBI 5 ms) and short-latency afferent inhibition (SAI 22 ms). Finally, we compared the effect of tonic and phasic voluntary contraction on SICICSPA3 and SICICSAP3.Using two superimposed TMS coils, CSAP produced little SICI at ISIs = 1 and 2 ms. However, at ISI = 3 ms, both CSAP and CSPA were equally effective at the same percent of maximum stimulator output. Despite this apparent similarity, combining SICICSPA3 or SICICSAP3 with other forms of inhibition led to quite different results: SICICSPA3 interacted in complex ways with CBI, SAI and SICICSPA2, whereas the effect of SICICSAP3 appeared to be quite independent of them. Although SICICSPA and SICICSAP were both reduced by the same amount during voluntary tonic contraction compared with rest, in a simple reaction time task SICICSAP was disinhibited much earlier following the imperative signal than SICICSPA.CSCSPA appears to activate a different inhibitory pathway to that activated by SICICSAP. The difference is behaviourally relevant since the pathways are controlled differently during volitional contraction. The results may explain some previous pathological data and open the possibility of testing whether these pathways are differentially recruited in a range of tasks.SICI •Opposite directions of conditioning stimulus (CS) used to suppress MEPs evoked by a conventional test stimulus.•Different directions of CS have different time courses of short-interval intracortical inhibition (SICI).•They also interact differently with short-latency afferent inhibition and with cerebellar inhibition.•They are differently affected in a reaction time task.•We suggest there are two forms of SICI in motor cortex. The latter evokes MEPs with longer latency and higher threshold /[amplitude of MEP by CBI only]. We used the same method to compute the change in SICICSPA3 (when CBI or SAI was presented). Normality was tested by the Shapiro-Wilk test. Paired t-tests were applied to compare SICICSPA3 with the change in SICICSPA3 when CBI or SAI was also performed.In experiments 3, 4 and 5, we compared the amount of inhibition produced by SICICSPA3 and SICICSAP3) as main factors were used to investigate changes in SICI in different brain states in each muscle (FDI and ADM). Then, we used a further two-way RM-ANOVA with factors “Orientation” (SICICSPA3 and SICICSAP3) and “Time”  to assess changes in SICI during the preparation of movement, separately in each muscle (FDI and ADM).In experiment 6, two separate two-way RM-ANOVAs with “Brain state”  and “Orientation”  in each experiment. There were no obvious violations of normality in these datasets. The critical value of significance was 0.05 in all statistics. Post hoc pairwise comparisons employed a Bonferroni correction.33.1AP, we extended the range of intensities for this orientation to 60–110% AMTAP. These two ranges are indicated in the graphs by dark and light symbols, respectively. The absolute intensities of the conditioning stimuli may appear to be quite high; this is because the conditioning coil was placed over the test coil which increased the distance between the scalp surface and the conditioning (D50) coil.This experiment investigated how SICI at ISIs = 2 and 3 ms varied with the intensity and orientation of the CS. PA (for both current directions). The x-axis plots CS intensity as a percent of AMTPA. At ISI = 3 ms , and a significant “Orientation x Intensity” interaction . Post hoc pairwise comparison showed that this was due to the fact that SICICSPA2 was stronger than SICICSAP2   , which was due to the fact that CSAP evoked inhibition at a lower threshold relative to the AMT of the CS than CSPA.The impression given by the graphs was borne out in the statistical analysis. In 110%AMT) C. In con= 0.768) D. In FigAP than CSPA, although it was approximately the same in terms of the relative AMT for each direction.To summarise, at ISI = 3 ms, the threshold for recruiting SICI was the same with both CS orientations, whereas at ISI = 2 ms, the absolute threshold was much higher for CS3.2PA), while we tested ISIs shorter and longer than in experiment 1. The data in PA was the lowest intensity that produced significant inhibition (one-tailed t-test compared to baseline) with both orientations of CS . At ISIs = 2, 3 ms, the results are very similar to those in experiment 1: both directions of CS produced the same amount of SICI at ISI = 3 ms, whereas SICI was absent at ISI = 2 ms for CSAP, whilst it was clear for CSPA. An even greater difference was seen at ISI = 1 ms, where there was strong inhibition for CSPA but much less for CSAP. Neither direction of CS produced inhibition at ISI = 5 ms.In this experiment, the intensity of the CS was the same for both directions ; post hoc pairwise comparison showed a stronger SICI with CSPA than CSAP at 1 ms (p < 0.001) and 2 ms (p = 0.020). Compared with baseline, there was a small amount of inhibition with CSAP1  but no detectable inhibition with CSAP2. In summary, the results confirm those of experiment 1  evoked approximately the same amount of SICI. However, CSPA3-SICICSPA2 evoked much greater inhibition than CSAP3-SICICSPA2. One way of comparing these values is to ask how much SICI we might expect to observe from combining the effects of two CS. The level of SICI evoked by CSAP3 and CSPA2 alone was 0.82 and 0.79, respectively. Thus if the effects were independent, we should expect that the result of combining them should be 0.82 ∗ 0.79 = 0.65. This prediction is similar to the observed value of 0.63 when CSAP3 and CSPA2 were combined experimentally (i.e. CSAP3-SICICSPA2). This contrasts with the combination of CSPA2 and CSPA3 (i.e. CSPA3-SICICSPA2). This combination produced SICI = 0.39 which was much more powerful than expected from independently combining SICICSPA3 (0.79) and SICICSPA2 (0.79 ∗ 0.79 = 0.62). Thus, rather than acting independently, CSPA2 and CSPA3 show temporal facilitation and produce more SICI than expected.PA3-SICICSPA2, SICICSPA3, and SICICSPA2. A one-way RM-ANOVA revealed a significant, global difference among them . Post hoc pairwise comparison showed that CSPA3-SICICSPA2 was significantly different from both SICICSPA3 (p < 0.001) and SICICSPA2 (p = 0.001). Similarly, another one-way RM-ANOVA on the level of SICI produced by CSAP3-SICIPA2, SICICSAP3, and SICICSPA2 revealed a significant difference among them . Post hoc pairwise comparison showed that CSAP3-SICIPA2 was different from both SICICSAP3 (p = 0.039) and SICICSPA2 (p = 0.035). Crucially, the two-way RM-ANOVA with “Orientation” (CSPA and CSAP) and “Condition”  and SICI at ISI of 3 ms in the presence of SICICSPA2 (CSPA3-SICICSPA2 and CSAP3-SICICSPA2)) as main factors revealed an “Orientation x Condition” interaction , which post hoc pairwise comparison showed was due to the fact that CSPA3-SICICSPA2 induced stronger cortical inhibition than CSAP3-SICIPA2 (p = 0.004).In the statistical analysis, we compared the level of inhibition produced by CS3.4AP and PA conditioning stimuli, we next tested whether these would interact in different ways with another input to M1.Since the results in experiment 3 suggested that there are differences in the inhibition produced by AP3 or CSPA3. CBI-CSPA3 yielded no greater inhibition than either alone, whereas CBI-CSAP3 produced more inhibition than either stimulus alone.CSAP3 and CBI-CSAP3. A one-way RM-ANOVA with Greenhouse-Geisser correction revealed a significant difference between them . Post hoc pairwise comparison showed that CBI-CSAP3 was significantly different from both SICICSAP3 (p = 0.003) and CBI (p = 0.027). However, there were no significant differences in another one-way ANOVA including CBI, SICICSPA3 and CBI-CSPA3 . Crucially, the two-way RM-ANOVA with “Orientation” (CSPA and CSAP) and “Condition”  as main factors revealed an “Orientation x Condition” interaction , which post hoc pairwise comparison showed was due to the fact that CBI-CSAP3 produced more inhibition than CBI-CSPA3 (p = 0.001). Interestingly, we also found that the calculated expected sum of CBI-CSAP3 (i.e. [CBI (0.73)] ∗ [SICICSAP3 (0.78)] = 0.58) was similar to the observed value (0.56).In the statistical analysis, we compared the level of inhibition evoked by CBI, SICI3.5As in experiment 4, we next examined how sensory afferent inputs to the motor cortex interact with SICI produced by different directions of CS.AP3 or CSPA3. SAI-CSPA3 yielded no greater inhibition than either alone, whereas SAI-CSAP3 appears to produce more inhibition than SICICSAP3.CSAP3 and SAI-CSAP3. The one-way RM-ANOVA revealed a significant difference among them . Post hoc pairwise comparison showed that SAI-CSAP3 was significantly different from SICICSAP3 (p = 0.01) but not different to SAI alone (p = 0.264). However, there were no significant differences in the one-way RM-ANOVA comparing SAI, SICICSPA3 and SAI-CSPA3 . Crucially, the two-way RM-ANOVA with “Orientation” (CSPA and CSAP) and “Condition”  as main factors revealed an “Orientation x Condition” interaction , which post hoc pairwise comparison showed was due to the fact that SAI-CSAP3 produced more inhibition than SAI-CSPA3 (p = 0.001). Again, the expected combination of SAI-CSAP3 (i.e. [SAI (0.62)] x [SICICSAP3 (0.74)] = 0.46) was not far from the observed value of 0.51.In the statistical analysis, we compared the level of inhibition evoked by SAI, SICICSPA3 when CBI was performed was calculated (1.36 ± 0.59). A paired t-test revealed that CBI suppressed SICICSPA3 significantly  or “Orientation x Brain state” interaction . However, there was a significant main effect of “Brain state” , indicating that contraction reduced SICI. Similar results were seen in ADM. A two-way RM-ANOVA with “Brain state” and “Orientation” as main factors revealed there was no effect of “Orientation”  or “Orientation x Brain state” interaction , but there was a significant effect of “Brain state”   A and B. 2,24 = 3.675, p = 0.041; ADM: F2,24 = 5.670, p = 0.001). Post hoc pairwise comparison revealed that SICICSAP3 decreased much earlier during the reaction time (RT35%) than SICICSPA3   C and D. ocks see .4PA). The present results suggest that an anterior-posterior conditioning stimulus (CSAP) of the same intensity can activate a different, and perhaps even independent, inhibitory circuit. We discuss the evidence for this conclusion in the following paragraphs.The present experiments explored the effect of reversing the direction of the conditioning stimulus (CS) on short-interval intracortical inhibition (SICI) at interstimulus intervals (ISIs) of 1–5 ms using a range of CS intensities. Conventionally, SICI is produced using a sub-motor threshold posterior-anterior (PA) conditioning stimulus . The problem with this is that AP and PA test stimuli evoke different combinations of I-waves which are differentially sensitive to SICI [Using different coils for the conditioning and test stimuli allows independent adjustment of the direction of CS and TS. This allowed us to maintain a conventional PA test stimulus while reversing the direction of the conditioning stimulus. Most previous investigations ,14,34,35A potential disadvantage of this method is that one coil (conditioning coil in the present experiments) rests on top of the other coil, which means that there is an additional distance from the scalp surface. This will reduce its focality and result in activation of a larger cortical area than if the coil were placed in contact with the scalp. However, this is unlikely to have had an important influence on the results since in the past SICI has been tested with a large number of different coil types of varying focality with little noticeable difference in outcome ,25.4.2PA and CSAP). This is perhaps unexpected since we usually express the intensity of a CS relative to its own AMT. Nevertheless, it is similar to the conclusion of Ziemann et al. [PA at ISI = 3 ms even though the CS intensity was the same in each direction. In contrast, at 2 ms, CSAP was much less effective than  CSPA. Hanajima [AP was less effective in evoking inhibition at 2 ms than 3 ms, although a direct comparison with the present data is difficult because both CS and TS were in the AP direction.Experiment 1 showed that at 3 ms, the absolute threshold for evoking SICI was approximately the same for both directions of CS  is usually 2–3 ms later than those evoked with a PA pulse (PA-MEPs), so maybe the onset of SICI evoked with CSAP is also delayed and begins at 3 ms. However, while this accounts for the difference in latency, it does not explain why the thresholds for CSAP and CSPA are approximately equal whereas the threshold for AP-MEPs is higher than for PA-MEPs. The answers may depend on precisely which population of neurons is activated by AP and PA pulses and their relative locations.The difference in the time course of SICI produced by CSAP that was approximately equal to 90% of the direction-appropriate AMT. This is the same relative intensity required when using a  CSPA. However, it is quite different to the behaviour at ISI = 3 ms, where the intensity of CSAP can be much lower than 90%AMTAP  threshold. The simplest explanation is that they both activate the same set of inhibitory interneurons, which are insensitive to the direction of the TMS pulse. Experiments 3–5 were conducted to test this. The rationale was that if CSI or CBI ,21,23, wPA could produce a SICI-like effect if the interval between them was 1–5 ms [Previous work showed that combining two  subthreshold CSs 1–5 ms . The auts 1–5 ms . Each CSPA stimuli (at 2 and 3 ms) produce an effect larger than the predicted sum of each stimulus alone. However, temporal facilitation was not evident when CSAP3 was paired with CSPA2. Importantly, the amount of SICI produced by CSAP3 alone was the same as when CSPA3 was used, and thus we might have expected to see the same amount of temporal summation with CSPA2. In fact, inhibition was only equal to the expected sum of the effect of each CS alone. This is consistent with the idea that CSPA and CSAP have an additive effect such as would be expected from activating two separate inhibitory circuits. This does not necessarily mean that they activate two different sets of inhibitory interneurons. It could be, for example, that excitatory inputs activated by CSPA and CSAP activate different fractions of the same inhibitory population, or that their inputs target different, non-interacting parts of the dendritic tree of a single neuron. Whatever the mechanistic details, the result suggests that inhibition by CSAP at 3 ms is not a time-delayed version of CSPA2.The results of experiment 3 were consistent with this idea of temporal facilitation since they showed that two CSNote that our CS intensities were above the threshold for producing some SICI and therefore slightly higher than in the original experiments of Bestmann et al. . However4.5CSPA2 was first examined by Daskalakis et al. [The interaction of CBI and SICIs et al. . As in tPA when measured in the presence of CBI  SICI has been investigated previously by Alle et al. and Udupa et al. [AP3. We conclude that the pathways responsible for SAI and SICICSPA3 interact with each other, whereas those responsible for SAI and SICICSAP3 are likely to be independent.These results and their interpretation are very similar to those for CBI. The combination of SAI and CSa et al. ,23. They4.7PA and CSAP probe different inhibitory circuits and that these circuits are modulated at specific times during the course of movement. The effects do not differ across the effector probed and are state-dependent, as no differences were found across rest or tonic activation. While speculative, changes in SICI probed with CSAP may reflect the influence of premotor area [PA, which may reflect direct involvement of M1 [The results of experiment 6 show for the first time that distinct SICI circuits are differentially modulated throughout the time course of movement preparation. The implication is, again, compatible with the hypothesis that CStor area ,38 whichnt of M1 ,6.What is activated by different directions of CS?1)The threshold for producing inhibition is the same in each direction when ISI is 3 ms or longer.2)PA compared with CSAP.Inhibition starts at shorter ISIs with CSThe results are consistent with the notion that opposite directions of CS activate two different populations of inhibitory neurons, but they give no information about where and what these neurons are. Nevertheless, they do give some clues.PA is caused by stimulation of synaptic terminals of basket cells that monosynaptically inhibit corticospinal neurons and/or activation of excitatory synapses onto the basket neurons  or neurons that inhibit excitatory inputs to corticospinal neurons (i.e. dis-facilitation) . Realistns as in .Fig. 9PoAP-TMS (single-pulse TMS with suprathreshold intensity in AP direction) shifts the site of neural activation anteriorly compared with PA-TMS (single-pulse TMS with suprathreshold intensity in PA direction). If CSAP activates at a more anterior site, it may discharge a cortico-cortical neuron that excites inhibitory neurons in the posterior location near to the corticospinal neurones. Because these inhibitory neurones interact with CBI and SAI in a different way to those activated by CSPA, they may represent a different population . I4.8AP3 and CSPA3 may be more similar, and it will be important to perform more studies in future in order to know if it is possible to generalize the conclusions. For example, by analogy with the latency difference between MEPs evoked by AP and PA test pulses, it is possible that inhibition using CSAP is a delayed version of CSPA. Thus, it could be argued that we should have compared CSAP3 with earlier timings of CSPA rather than CSPA3. Udupa et al. [PA2 and CSPA3 interacted with SAI, but such effects would not be sufficient to explain the very different interactions we saw here.The experiments used a limited range of stimulus intensities and interstimulus intervals. Thus, our conclusions are also limited to the parameters we have investigated. It is possible that, with other parameters, the effects of CSa et al.  found thAP conditioning stimulus to suppress a PA test response (AP direction), which again is much higher than the range of CS intensities we used in the experiments.A second limitation of the present study was the potential contamination of SICI at ISI = 3 ms by short-interval intracortical facilitation (SICF). Using two pulses of the same direction, the second peak of SICF occurs at around 2.8 ms , and a sresponse B, reduceAP conditioning stimuli differ between individuals, which probably indicates that it is not possible with a TMS pulse to isolate completely one set of inhibitory neurones from another.The experiments were also limited technically by the fact that we could not randomize the direction of the CS from trial to trial and had to use two separate, overlapping coils, rather than a single one. Achieving these would probably have reduced the variability of the results, but we think it unlikely to change the main conclusions. Finally, we note that the differences between PA and 5We show that different directions of TMS-induced currents in the brain are capable of recruiting two independent sets of inhibitory inputs over a range of ISIs used to probe SICI. Po-Yu Fong: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. Danny Spampinato: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. Lorenzo Rocchi: Conceptualization, Methodology, Writing – review & editing. Ricci Hannah: Conceptualization, Methodology, Writing – review & editing. Yinghui Teng: Writing – original draft, Investigation. Alessandro Di Santo: Investigation. Mohamed Shoura: Investigation. Kailash Bhatia: Conceptualization, Resources, Supervision. John C. Rothwell: Conceptualization, Methodology, Resources, Writing – review & editing, Supervision, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The existing literature on internal branding has often adopted a managerial-based approach and seldom considered employees’ perceptions. Therefore, there is a need to understand the perspective of frontline and non-managerial employees. In this context, the current study investigates the impact of internal brand management on brand commitment, brand citizenship behavior, and sustainable competitive advantage for the hotel industry. A survey-based quantitative data was gathered from 390 non-managerial frontline staff working in 3-, 4-, and 5-star hotels of Pakistan. The results revealed that internal brand management positively impacts brand commitment, brand citizenship behavior, and sustainable competitive advantage. Besides, brand commitment has a positive impact on brand citizenship behavior and sustainable competitive advantage. Moreover, brand citizenship behavior has a positive impact on sustainable competitive advantage. In addition, the mediating roles of brand commitment and brand citizenship behavior exist between internal brand management and sustainable competitive advantage. The research implications, together with research limitations, have also been discussed. Branding has proved to be the best trend abounding the hotel industry worldwide –3. In coThus, hotels hire human and physical resources to meet their targets , and keeBrand Commitment and Brand citizenship behavior are often the results of internal brand management, and both provide the sources of sustainable competitive advantage. Brand commitment is a psychological and emotional attachment of an employee to the brand he/she is working for. It is a crucial driver for the success of many industries, especially the hotel industry . In prevFurthermore, the previous empirical studies , 20 onlyTherefore, this study aims to utilize the concept of internal branding to create a link between marketing and human resources. In this context, the effect of internal brand management on brand commitment, brand citizenship behavior, and sustainable competitive advantage has been examined for the hotel industry. The expected outcomes of this will be significant, as the recent researchers , 25–27 sThe current study results would carry the importance of internal branding in the hotel industry and reflect the process through which it leads toward achieving sustainable competitive advantage. Moreover, it will highlight the role of brand commitment and brand citizenship behavior in achieving sustainable competitive advantage. Furthermore, it emphasizes that in this era of intense competition, the hotel industry needs to attain sustainable competitive advantage through internal branding focused on their unique, rare, and inimitable employees.The rest of the sections have been structured as follows. The relevant literature has been presented in the next section. After that, hypotheses have been formulated, followed by the portrayal of methods. After that, the data were analyzed, and the results were discussed. Eventually, both theoretic and applied implications have been presented.Strategic managers have always focused on achieving sustainable competitive advantage, especially in highly competitive markets . AccordiSimilarly, internal brand management can be defined as a strategy, or a systematically organized process, which leads the employees toward an acceptable brand behavior . AccordiLikewise, brand commitment is a "psychological attachment of employees to their organization, which motivates them to put an extra effort to achieve the organizational goals" . Brand cBrand citizenship behavior is a voluntary and unofficial behavior of the employees, strengthening the brand . This coThe resource-based theory supports the relationship between internal branding and sustainable competitive advantage, while social exchange theory explains the relationship between internal branding, brand commitment, and brand citizenship behavior. Both approaches are related to human resources and employees because the resource-based view portrays a firm’s competitiveness resulting from its resources and links these resources with people . Social A total commitment is required to make a brand unique and thriving across all the employees of an organization , 45. 4949 suggesPrevious researchers , 55 obseInvesting in leadership development, primarily transformational leadership, is worthwhile and essential to promote employees’ volitional and positive work behaviors, such as organizational citizenship behavior . Transfo1: Internal brand management positively influences brand commitmentH noted a So, there is a consensus in the primary literature of organizational behavior , 65 and 2: Brand commitment positively influences brand citizenship behaviorHBoth researchers and practitioners agree that effective brand development is needed to precede a competitive advantage in the hotel industry. In contrast to product brands, customer perception of the hotel service type is dependent on the service delivery performance  due to dTo facilitate brand performance, the role of internal branding depends on the perception that an employee’s representative resources  can be used to provide an ongoing competitive advantage for the organization . Thus, b3: Brand citizenship behavior positively influences sustainable competitive advantageH suggeste4: Internal brand management positively influences brand citizenship behaviorHCompliance with internal branding is the key to achieve a sustainable competitive advantage; for example, a motivating leader positively influences the internal branding through seeking to solicit knowledge and learning by creating an environment conducive to working conditions, promoting psychological and personal attention, creating ingenuity, and encouraging problem-solving decisions . A focus5: Internal brand management positively influences sustainable competitive advantageHAccording to , 49, a nBrand commitment reflects the equal capacity for the psychological identification of an employee and his or her involvement with the organization . In addi6: Brand commitment positively influences brand sustainable competitive advantageHWhen employees have understandable brand values, they engage with the brand psychologically and emotionally . Brand c7: Brand commitment mediates between internal brand management and brand citizenship behavior.HBrand citizenship behavior is a potential consequence of internal brand management . The sucFurthermore, it has been examined that brand citizenship behavior partially mediates the relationship between franchisor support and franchisee-perceived brand image . The mos8: Brand citizenship behavior mediates the relationship between brand commitment and sustainable competitive advantage.H noted thUpon these mentioned propositions, the Hierarchy-of-Effects model can be utilized to understand the cascading effect. Brand commitment and brand citizenship behavior represent the employee’s internal process resulting from employees’ evaluation of the internal brand management, leading to the achievement of the firm’s sustainable competitive advantage. In other words, internal brand management of the hotels is unlikely to influence the sustainable competitive advantage unless employees’ clear brand commitment and brand citizenship behavior occur. Consequently, there is a likely case to consider brand commitment and brand citizenship behavior as a combined mediator between internal brand management and sustainable competitive advantage. Based on the given theoretical and empirical support, it is hypothesized:9: Brand commitment and brand citizenship behavior sequentially mediate the impact of internal brand management on sustainable competitive advantage.HThe hypothesized relationships have been depicted in Since the research objective was to examine the impact of internal brand management factors on sustainable competitive advantage, primary data from the participants were collected through a structured questionnaire using a quantitative approach. The respondents of this study were informed about the purpose of data collection. It was ensured that the collected data would be kept confidential and only be used for this research. The properly structured consent forms were distributed enclosed by envelope along with the questionnaire.The population for this study was the non-managerial or frontline staff working in 3-, 4- and 5-star hotels of Pakistan. The frontline staff is usually in direct contact with customers, known as customer-interface employees, so it is essential to understand their perspective about internal branding and competitive advantage . Data weFurthermore, purposive sampling was chosen to focus on people with particular characteristics compatible with the research purpose . Four huSince the data were collected from non-managerial staff, the education level was comparatively less, as 74.1% of respondents were intermediate. According to Ethical review and approval were obtained for this study on human participants from the committee named as ‘Ethical Research Committee’ of the Institution (NML-ERC/2020-014). Moreover, the participants provided their written informed consent to participate in this study.According to , internaInitially , introduA hotel’s competitive advantage is referred to as better services and performance than its competitors in the market . In thisThe mean value shows the center of data as many statistical analysts consider it a standard measure of central distribution of data. The mean value of IBM (3.89), BC (3.36), BCB (4.09), and SCA (-3.75) show the central distribution of data for these respective variables.The measurement model is used to assess errors in evaluating the theoretical concepts. Confirmatory Factor Analysis (CFA) was used to analyze the factor structure of the model. The estimation method maximum likelihood (ML) was used to check the loading of items on the corresponding factors, as shown in 2/df = 2.737<3 indicates a well-fitted model. Similarly, the value of SRMR = .0372 is within the acceptable range (0 <SRMR < .08) [Different model fit indices were used to check how data fit the theoretical model. The ratio of chi-square to its degree of freedom (CMIN/DF) was computed as an essential criterion of model fitness. The ratio of χR < .08) , 86. WheR < .08) , 88. TheR < .08) . In addiR < .08) , 90.The construct validity was established through convergent and discriminant validities. The convergent validity ensures whether the items load on the corresponding factors. On the other hand, discriminant validity measures how the items are different from other factors. The average variance extracted (AVE) values in Furthermore, the value of Cronbach’s alpha reflects internal consistency (reliability). It measures how closely the items of a group are related to each other.  stated tAfter confirming construct validity and reliability, the structural model was analyzed to test the hypotheses, as depicted in 1, H2, and H3 are concerning the direct relationships of adjoining variables. The results  demonstrate that these hypotheses have been accepted related to variables of internal branding, brand commitment, brand citizenship behavior, and competitive advantage. Similarly, H4, H5, and H6 are related to the direct relationships between variables bypassing the adjoining variables. The findings  confirm these hypotheses. The summary of details is presented in H7, H8, and H9, have been accepted , indicating the partial mediations. The significance of these relationships was computed by applying the bootstrapping procedure in AMOS [Likewise, hypotheses related to mediation effects, i.e., H in AMOS .This research examines the impact of internal branding on brand commitment, brand citizenship behavior, and sustainable competitive advantage. It has been observed that internal branding is a strong predictor of brand commitment. The relationship between both variables is significant and positive . The results are consistent with previous studies such as , 70. In Similarly, the results showed a positive relationship between brand commitment and brand citizenship behavior . This relationship has also been reported positive in previous research , 62. TheThe relationship between internal branding and sustainable competitive advantage shows a strong, positive, and significant relationship . Though the previous research theoretically discussed that internal branding is a potential route to acquiring sustainable competitive advantage , the curRegarding the unique contributions of the current study, the results confirm brand commitment as a significant mediator between internal branding and brand citizenship behavior. Similarly, the mediating role of brand citizenship behavior between brand commitment and sustainable competitive advantage has been tested. The results showed brand citizenship behavior as a significant mediator between brand commitment and sustainable competitive advantage. In previous research, brand citizenship has been considered a mediator between brand commitment and brand performance and internal branding and customer satisfaction. In both studies, brand citizenship behavior has been proved as a positive and significant mediator , 79, 95.Organizations dealing with generic products or services do not focus on the internal branding of employees but on price competition. But since the hotel industry has the potential to provide differentiated services, it can get the advantage of sustainable competitiveness through internal branding to earn above-average profits. However, there is very little knowledge available on internal branding in the literature, and, furthermore, this concept requires empirical evidence to confirm the consequences of internal branding. In this regard, the current study contributes to the literature by providing empirical evidence for the relationships between the variables not studied before, such as internal brand management (IBM) and sustainable competitive advantage (SCA) in a hotel setting. Moreover, brand commitment (BC) and brand citizenship behavior (BCB) as sequential mediators, integrating them between IBM and SCA, have also been discussed and empirically proved, which were not tested in previous research. In other words, internal branding not only contributes to sustainable competitive advantage directly but also stimulates brand commitment and brand citizenship behavior of hotel employees, which in turn help establish SCA. Hence, IBM, BC and BCB are the fundamental constituents of SCA. recommenFurthermore, previous studies on managerial staff, capital, machinery, and monetary assets have been taken as a sustainable competitive advantage. In contrast, this study followed the different parameters  to measuSimilarly, in previous research, there was an argument that whether the brand commitment is uni-dimensional (affective commitment only) or multidimensional . By following the recommendations of , this stThe findings of the study provide managerial insights to hoteliers to create and sustain a competitive advantage. The sustainable competitive advantage of functions is heavily dependent on intangible strategic assets such as service employees. Internal brand management is particularly relevant in the hospitality industry due to the inconsistent nature of demand and service quality in the hospitality industry. A company’s competitive advantage helps gain more profits from the shareholders. Similarly, creating a sustainable competitive advantage is the most crucial goal of any organization and perhaps the most critical quality that requires the attention of any organization. This study showed how internal brand activities could help brands gain sustainable competitive advantage. The organizations that emphasize their employees’ internal branding stimulates brand commitment and brand citizenship behavior to generate sustainable competitive advantage ultimately.Hotel managers should consider non-managerial frontline staff valuable internal stakeholders. Internal branding cannot be thought of in isolation, for example, at the time of recruitment, the employees who have fit with internal branding programs should be hired. Marketers and HR department need to work on closely the internal brand management system through daily briefing, group meetings, internal newsletters, notice boards, videos, blogs and training & development to achieve better consequences of IBM. Furthermore, close coordination among employees would create a sense of ownership to strengthen team spirit and internal branding. Moreover, managers should measure perceptions of brand and behavior of employees through continual in-house market research and take customized remedial actions after dialectical inquiry to reconcile brand perception of their employees .This research study has focused on competitive advantage driven through employees and HR of firm/brand. This research study has identified the factors that cause employees to be involved in brand citizenship behavior and its impact on sustainable competitive advantage. The study demonstrated that the hotels must focus on the frontline non-managerial staff and ensure that the communication channels effectively communicate. Usually, low-level and non-managerial staff in organizations is not considered worthy of communication, but the results of this study reveal that these employees appreciate the contact and like to be well informed about the standards and values.Another important factor of internal branding highlighted by this study is transformational leadership. This is the most appreciated factor by non-managerial staff . A superIn this era of intense competition, organizations need to look up new strategies to pursue sustainable competitive advantage. In the hotel industry, customer’s perception is based on the services they experience. Before making their positive perception about their experience at a hotel, it is essential to create a positive perception of those who deliver the services and create the perception of guests/customers. Since human resources play a vital role in providing services, the service industry should think differently about competitive advantage. Branding toward employees is as much crucial as branding focused on customers. According to the statistical results obtained in this research, it is clear that there is a significant and positive relationship between internal branding and sustainable competitive advantage. Brand commitment and brand citizenship behavior act as the critical mediators between internal branding and sustainable competitive advantage. The literature and the empirical evidence show that internal branding leads toward achieving sustainable competitive advantage through brand commitment and brand citizenship behavior.There are a few limitations of this study. The first limitation is that it is quantitative research in nature. However, it would be interesting for future researchers to qualitatively investigate the perception of frontline employees about internal branding because more and different internal branding factors can be observed with this approach. The second limitation is that the purposive sampling method used in this research has a generalizability issue. Future researchers may adopt different sampling techniques to collect data. The third limitation is that the present analysis is not comparative. However, future researchers may conduct a comparative study to compare the perception of managers and their subordinates about internal branding and brand citizenship behavior, and the comparison can also be performed among 3-, 4- and 5-star hotels. The fourth limitation is that only a few constructs have been taken as antecedents and the consequences of internal branding. Future studies should investigate other possible outcomes and relate them to customer-related values such as customer loyalty and customer satisfaction.S1 Dataset(RAR)Click here for additional data file.


Aspergillus Niger, and Aspergillus Flavus, on a nanosilver colloid solution. The antibiosis is compared with a standard silver ion solution at the same concentration as in the experimental process. This study proved that the nanosilver colloid prepared by the electrical spark discharge method (ESDM) is free of any chemical additives, has a microbial control effect, and that the effect is much better than the Ag+ standard solution at the same concentration. 3M Count Plate (YM) is used to test and observe the colony counts. The microbial control test for yeast, Aspergillus Niger, and Aspergillus Flavus is implemented in the nanosilver colloid. In addition to Aspergillus flavus, an Ag+ concentration of 16 ppm is enough to inhibit the growth of the samples. At the same concentration, the nanosilver colloid has a much better microbial control effect than the Ag+ standard solution, which may be because the nanoparticle can release Ag+ continuously, so the solution using the ESDM has a more significant microbial control effect.This is a study of an antimicrobial test, including yeast,  Aspergillus is the main fungal contaminant and one of the important factors leading to the decay and accumulation of mycotoxins in food. The pollution of these microorganisms will not only cause huge economic damages and losses, but also pose a threat to human health. Therefore, there is an urgent need to develop safe reagents to control bacterial and fungal contamination. Due to developing technologies, nanotechnology can be one of the ways to prevent food decay. We are surrounded by the antimicrobial applications of nanosilver, in our daily necessities, textile clothing, and cosmetics, as well as in our air-conditioners and washing machines [Food and water are indispensable substances in human life. However, many foods become inedible or difficult to preserve due to the growth of microorganisms. Contamination by bacteria and fungi can cause a decrease in the freshness of food and cause an unpleasant taste, which can damage the food. machines . When namachines ,3. The Fmachines ,5,6. Undmachines ,8,9.The current so-called bactericidal mechanism of silver is that when nanosilver is close to microorganisms, it will cleave the protease that metabolizes oxygen. After losing their effectiveness, they cannot produce normal metabolism for oxygen, leading to their natural death, and finally they will be excreted from the body with the body’s metabolism. This is different from general bactericidal drugs, which also kill the good bacteria in the body. After nanosilver leaves the body, the cells remain intact, so nanosilver is safe for humans, reptiles, plants, and other multicellular organisms.Aspergillus niger, and Aspergillus flavus resistance.This study used the electrical spark discharge method (ESDM) to prepare nanosilver colloids and obtained good research findings. As the nanosilver colloid that is produced can release silver ions ,11,12, aThe architecture of the nanometer metallic colloid preparation system is shown in on is the time of conduction when the electrodes will successfully carry out arc discharge, Toff is the time of stop, and after a successful electrode discharge, the DW should be restored to the state of insulation in order to facilitate the discharge of the next cycle and to eliminate the metallic particles [The ESDM system can use an oscillator to observe the voltage and current wavelengths between the electrodes, as shown in articles .ON-TOFF was adjusted to 50–50 µs, and preparation time was performed for 40 min. The electrodes were weight before and after the preparation, and the results are shown in The parameter settings of the ESDM are described as follows. The 99.99% pure Ag was selected as electrodes. The two electrodes were cleaned with DW and fixed in the ESDM and beaker, and the upper and lower electrodes were aligned. T+ and 300 ppm nanoparticle), as samples for microbial control testing. No. 2 was a 32 ppm Ag+ standard solution.Preparation of testing solutions: the nanosilver colloid was prepared by ESDM  was used, one loop of fungi was scraped and put in 25 mL, 0.05% Tween80 solution to make the test solution, and the concentration was adjusted to 103M Petrifilm™ Yeast and Mold Count Plate (petrifilm) was used for microbial control testing ,21: the The experimental process was approximately divided into two parts; one was the preparation of the nanosilver colloid and the strain activation and cultivation, while the other was the microbial control cross testing of the prepared test solution and strain. It is described in the following four steps:The No. 1 solution used ESDM to prepare the nanosilver colloid, and the related parameter settings and characteristics are described. UV–Visible Spectroscopy  uses visible light and UV tubes as a light source. Nanoparticles have surface plasmon resonance (SPR) in light . DiffereThe laser light scatterometer  uses non-invasive back scattering (NIBS) to detect a scattered light source ,24. The Aspergillus niger, and Aspergillus flavus. The Aspergillus niger and Aspergillus flavus were pure strains bought from BCRC (Bioresource Collection and Research Center) [There were three sample types: yeast,  Center) , while tAspergillus niger and Aspergillus flavus are described as follows:Aspergillus niger and Aspergillus flavus drying tubes. The inner tube Aspergillus were washed off, and the 0.1 mL bacteria solution suspension was instilled in the center of solid medium. The culture medium for Aspergillus niger was malt extract agar (MEA), while that for Aspergillus flavus was potato dextrose agar (PDA), which were uniformly smeared by an L-shaped glass rod, placed in an incubator at the ambient temperature of 25 °C, and cultured for 7–9 days.Cultivation of the first-generation species: 0.3–0.5 mL specified liquid medium was dripped into the unsealed 2 was scraped and uniformly smeared by an L-shaped glass rod, and cultivated in the incubator until the mold spores grew. The experiment was conducted on a sterile console.Cultivation of the second-generation species: The cultivation of the second-generation species is also known as secondary offspring cultivation, where uncontaminated and complete colonies were selected from the culture medium of the first-generation species, about 0.5 cmThe activation procedures of The loop was used to scrape the cultured strain off the culture dish, it was soaked in a 0.05%, 25 mL Tween 80 solution, and the solution was diluted 100× and 10,000×, respectively.A pipette was used to extract 0.1 mL of the solution diluted 100×, which was placed under a digital microscope. The concentration was observed and adjusted with DI water, and the number of strains was smaller than 600 in order to maintain the optimum observed number of strains on the count plate below 300.The No. 1 and No. 2 test solutions were mixed with quantitative bacteria solution to prepare the required concentration.The pipette was used to extract 1 mL of the prepared solution, which was injected onto the count plate, covered with the membrane, flattened by a press plate, placed in the incubator, and observed regularly.Petrifilm was used to test and observe the colony counts, and it involved the following:RP: residual amount of the number of colonies by percentage;T: total amount of colonies in 2 ppm concentration; andR: residual amount of the number of colonies.The symbol explanation is shown in Aspergillus niger is shown in Aspergillus flavus is shown in The concentrations of the nanosilver colloid and ion silver solutions were tested using a count plate, and the test results are described in Aspergillus niger, and Aspergillus flavus. Using the 3M Count Plate can easily achieve the counting results. In the Aspergillus niger test, the nanosilver colloid had more of an effect even though there was some deviation of the test. The 16 ppm nAg and Ag showed no A. niger on the colony count plate. In the Aspergillus flavus test, under the same concentration, the nanosilver colloid had a better microbial control effect. Even with 32 ppm colloid and Ag+, the A. flavus still grew but a concentration of 16 ppm can inhibit the growth. In the yeast test, 8 ppm of the nanosilver colloid prevented the yeast from growing.By using the ESDM, the nanosilver colloid could be prepared. This method is costless, simple, and free of chemical agents. In this study, the method applied in synthesizing nanosilver colloid allowed antimicrobial activity, including yeast, Aspergillus is a ubiquitous fungus that grows on stored grains. Toxins produced by some species can harm human and animal health and cause liver and kidney toxicity, immunosuppression, and carcinogenicity. The main fungicides used to prevent the growth of fungi may be toxic to humans and their repeated use will increase the resistance of microorganisms over time. Nanosilver has antimicrobial results, including yeast, Aspergillus niger, and Aspergillus flavus. By using the 3M Count Plate, the results were easily achieved. The results of the microbial control test by count plate are described as follows (see Compared with the number of colonies with a 2 ppm concentration, the higher the concentration, the lower the amount of residual colonies.Aspergillus flavus, the nanosilver colloid had a better microbial control effect than Ag+, at the same concentration.With addition to Aspergillus flavus, all strains had a 32 ppm concentration. The residual RP was less than 0.1%.With addition to Aspergillus niger > Aspergillus flavus.Effect of the microbial control of nano silver and silver ions, in order, was: yeast > lows see :ComparedAspergillus niger, Aspergillus flavus, and yeast. The results are summarized as follows:The nanosilver colloid was prepared using the electrical spark discharge method, which is free of any chemical additives. The absorption was around 1.13 at the wavelength of 390 nm and the average zeta potential was −22.3 mV ± 4.77 mV. With the result of the SEM, the particle size was approximately 30 nm in diameter.+ standard solution, at the same concentration.The nanosilver colloid prepared by ESDM had a microbial control effect, and the effect was much better than the Ag+ concentration of 16 ppm was enough to inhibit the growth of Aspergillus niger, Aspergillus flavus, and yeast. The antimicrobial effect of nanosilver and Ag+ was, in order of strength, yeast > Aspergillus niger > Aspergillus flavus.Among all the test samples, the Ag+ standard solution because the nanoparticle can release Ag+ continuously, such that the No. 1 test solution had a more significant microbial control effect.At the same concentration, the nanosilver colloid had a much better microbial control effect than the AgA microbial control test was conducted with nanosilver colloids on