Human Salivary Alpha-amylase Research Articles

Neuroprotection against Aluminum Chloride-Induced Hippocampus Damage in Albino Wistar Rats by Leucophyllum frutescens (Berl.) I.M. Johnst. Leaf Extracts: A Detailed Insight into Phytochemical Analysis and Antioxidant and Enzyme Inhibition Assays.

A previously unstudied medicinal plant, Leucophyllum frutescens (Berland.) I.M. Johnst. (Scrophulariaceae) was investigated to evaluate its potential in preventing and treating neurodegenerative diseases, including Alzheimer's disease. Methanolic leaf extract (MELE) and its fractions (HELE, CHLE, and BULE) were evaluated for their polyphenolic content and antioxidant activity by five different methods, including in vitro enzyme inhibition assays, which are clinically linked to neurodegenerative diseases. The potentially active n-butanol fraction (BULE) was further evaluated for its neuroprotective effects using an albino rat animal model and phytoconstituents profiling using Liquid chromatography with tandem mass spectrometry (LC-MS/MS), and in silico molecular docking by Maestro® Schrödinger. The n-butanol fraction (BULE) in the hydroalcoholic leaf extract exhibited the highest total phenolic content (230.435 ± 1.575 mg gallic acid equivalent gm-1± SD). The chloroform leaf extract exhibited the highest total flavonoid content (293.343 ± 3.756 mg quercetin equivalent gm-1± SD) as well as the highest antioxidant content, which was equivalent to Trolox, with five assay methods. Similarly, the chloroform and n-butanol fractions from the hydroalcoholic leaf extract significantly inhibited human acetylcholinesterase and butyrylcholinesterase with their IC50 values of 12.14 ± 0.85 and 129.73 ± 1.14 µg∙mL-1, respectively. The in vivo study revealed that BULE exhibited a significant neuroprotective effect at doses of 200 and 400 mg/kg/day in an aluminum chloride-induced neurodegenerative albino rat model. The LC-MS/MS analysis of BULE tentatively confirmed the presence of biologically active secondary metabolites, such as theobromine, propyl gallate, quercetin-3-O-glucoside, myricetin-3-acetylrhamnoside, isoquercitrin-6'-O-malonate, diosmetin-7-O-glucuronide-3'-O-pentose, pinoresinol diglucoside, asarinin, eridictoyl, epigallocatechin, methyl gallate derivative, and eudesmin. The results from the computational molecular docking of the identified secondary metabolites revealed that diosmetin-7-O-glucuronide-3'-O-pentose had the highest binding affinity to human butyrylcholinesterase, while isoquercetin-6'-O-malonate had the highest to human acetylcholinesterase, and pinoresinol diglucoside to human salivary alpha-amylase. The present study concluded a need for further exploration into this medicinal plant, including the isolation of the bioactive compounds responsible for its neuroprotective effects.

High-Throughput Sequencing Analysis of the Changes in the Salivary Microbiota of Hungarian Young and Adult Subpopulation by an Anthocyanin Chewing Gum and Toothbrush Change.

The sour cherry contains anthocyanins, which have bactericide action against some oral bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa). Sour cherry also has antibiofilm action against Streptococcus mutans, Candida albicans, and Fusobacterium nucleatum. Our earlier research proved that chewing sour cherry anthocyanin gum significantly reduces the amount of human salivary alpha-amylase and Streptococcus mutans levels. The microbiota of a toothbrush affects oral health and regular toothbrush change is recommended. A total of 20 healthy participants were selected for the study. We analysed saliva samples with 16S rRNA sequencing to investigate the effect of 2 weeks (daily three times, after main meals) of chewing sour cherry anthocyanin gum-supplemented by toothbrush change in half of our case-control study cohort-after scaling on human oral microbiota. A more stable and diverse microbiome could be observed after scaling by the anthocyanin gum. Significant differences between groups (NBR: not toothbrush changing; BR: toothbrush changing) were evaluated by log2 proportion analysis of the most abundant family and genera. The analysis showed that lower level of some Gram-negative anaerobic (Prevotella melaninogenica, Porphyromonas pasteri, Fusobacterium nucleatum subsp. vincentii) and Gram-positive (Rothia mucilaginosa) bacteria could be observed in the case group (BR), accompanied by build-up of health-associated Streptococcal network connections.

Inhibition mechanism of alpha-amylase, a diabetes target, by a steroidal pregnane and pregnane glycosides derived from Gongronema latifolium Benth.

Alpha-amylase is widely exploited as a drug target for preventing postprandial hyperglycemia in diabetes and other metabolic diseases. Inhibition of this enzyme by plant-derived pregnanes is not fully understood. Herein, we used in vitro, in silico, and in vivo studies to provide further insights into the alpha-amylase inhibitory potential of selected pregnane-rich chromatographic fractions and four steroidal pregnane phytochemicals (SPPs), viz: marsectohexol (P1), 3-O-[6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→14)-β-D-oleandropyranosyl]-11,12-di-O-tigloyl-17β-marsdenin (P2), 3-O-[6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl]-17β-marsdenin (P3), and 3-O-[6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-canaropyranosyl]-17β-marsdenin (P4) derived from Gongronema latifolium Benth. The results revealed that the SPPs source pregnane-rich chromatographic fractions and the SPPs (P1–P4) exhibited inhibitory potential against porcine pancreatic alpha-amylase in vitro. Compounds P1 and P2 with IC50 values 10.01 and 12.10 µM, respectively, showed greater inhibitory potential than the reference acarbose (IC50 = 13.47 µM). Molecular docking analysis suggests that the SPPs had a strong binding affinity to porcine pancreatic alpha-amylase (PPA), human pancreatic alpha-amylase (HPA), and human salivary alpha-amylase (HSA), interacting with the key active site residues through an array of hydrophobic interactions and hydrogen bonds. The strong interactions of the SPPs with Glu233 and Asp300 residues may disrupt their roles in the acid-base catalytic mechanism and proper orientation of the polymeric substrates, respectively. The interactions with human pancreatic amylase were maintained in a dynamic environment as indicated by the root mean square deviation, radius of gyration, surface accessible surface area, and number of hydrogen bonds computed from the trajectories obtained from a 100-ns molecular dynamics simulation. Key loop regions of HPA that contribute to substrate binding exhibited flexibility and interaction potential toward the compounds as indicated by the root mean square fluctuation. Furthermore, P1 significantly reduced blood glucose levels and area under the curve in albino rats which were orally challenged with starch. Therefore, Gongronema latifolium and its constituent SPPs may be exploited as inhibitors of pancreatic alpha-amylase as an oral policy for impeding postprandial blood glucose rise.

Investigation of effective natural inhibitors for starch hydrolysing enzymes from Simaroubaceae plants by molecular docking analysis and comparison with in-vitro studies

The present study aims to find the effective natural enzyme inhibitors against alpha-amylase and alpha-glucosidase from the array of compounds identified in plants of the Simaroubaceae family using molecular docking and ADME/Toxicity studies. Among the 218 compounds docked against seven enzymes, buddlenol-A and citrusin-B showed the best binding energies (kcal/mol) of -7.830 and -7.383 against human salivary alpha-amylase and pancreatic alpha-amylase respectively. The other two compounds 9-hydroxycanthin-6-one and bruceolline-B had the best binding energy of -6.461 and -7.576 against N-terminal and C-terminal maltase glucoamylase respectively. Whereas the binding energy of prosopine (-6.499) and fisetinidol (-7.575) was considered as the best against N-terminal and C-terminal sucrase-isomaltase respectively. Picrasidine-X showed the best binding energy (-7.592) against yeast alpha-glucosidase. The study revealed that the seven compounds which showed the best binding energy against respective enzymes are considered as the ‘lead hit compounds’. Even though the ‘lead hit compounds’ are not obeying all the laws of ADMET, the drug-likeness properties of 9-hydroxycanthin-6-one, fisetinidol, picrasidine-X, and prosopine were considerable. Also, kaempferol-3-O-pentoside was the recent compound identified from the Simarouba glauca plant extract found to be one among the top five lead hit compounds against four enzymes. This study provides valuable insight into the direction of developing natural compounds as potential starch hydrolysing enzyme inhibitors for managing type 2 diabetes.

Immunosensor Based on Zinc Oxide Nanocrystals Decorated with Copper for the Electrochemical Detection of Human Salivary Alpha-Amylase.

(1) Background: Nanocrystals (NCs)-based electrochemical sensors have been proposed for biomarkers detection, although immunosensors using ZnO NCs decorated with copper are still scarce. (2) Methods: Electrochemical immunodetection of human salivary alpha-amylase (HSA) used ZnO, CuO, and ZnO:xCu (x = 0.1, 0.4, 1.0, 4.0, and 12.0) NCs. (3) Results: Substitutional incorporation of Cu2+ in the crystalline structure of ZnO and formation of nanocomposite were demonstrated by characterization. Graphite electrodes were used and the electrochemical signal increased by 40% when using ZnO:1Cu and 4Cu (0.25 mg·mL−1), in an immunosensor (0.372 mg·mL−1 of anti-alpha-amylase and 1% of casein). Different interactions of HSA with the alpha-amylase antibody were registered when adding the NCs together, either before or after the addition of saliva (4 μL). The immunosensor changed specificity due to the interaction of copper. The ZnO:1Cu and ZnO:4Cu samples showed 50% interference in detection when used before the addition of saliva. The immunosensor showed 100% specificity and a sensitivity of 0.00196 U·mL−1. (4) Conclusions: Results showed that the order of NCs addition in the sensors should be tested and evaluated to avoid misinterpretation in detection and to enable advances in the validation of the immunosensor.

The Potency of Cinnamon as An Anti-Diabetic and Anti-Covid19 based on Its Mineral Content and Phenolic Compounds

The purpose of this study were to determine the mineral content (Cr, K and Ca) as well as total phenolic (TPC) and total flavonoids (TFC) in cinnamon, then to know the potency of some phenolic compounds in cinnamon as alpha amylase inhibitors and its binding activity to S protein of covid19. The mineral content was determined by the ICP method, while the TPC and TFC levels were determined spectrophotometrically. In silico analysis was carried out by docking techniques using human salivary alpha amylase and S covid19 protein as receptors and several polyphenol compounds in cinnamon as ligands. The results have shown that the levels of Cr, K, Ca, TPC and TFC of cinnamon, were 0.524 ppm, 4033ppm, 17453 ppm, 1.55% and 4.26%, respectively. The results of in silico analysis has shown that kaempferol 3-O-glucoside and quercetin in cinnamon are able to bind to human saliva α-Amylase on its active site with a binding affinity that are relatively the same as acarbose has. The docking analysis also has shown that kaempferol, quercetin and rutin of cinnamon were able to bind to the Receptor Binding Domain (RBD) of S protein of covid19, so that these compounds also have the potency to be anticovid19.

In Silico and In Vitro Studies of the Inhibitory Effect of Antihistamine Drug Cyproheptadine Hydrochloride on Human Salivary Alpha Amylase.

For the first time, the inhibitory effects on the human salivary alpha-amylase activity of the anti-inflammatory drugs indomethacin, diclofenac sodium, ketoprofen, diclofenac potassium, diclofenac, triamcinolone acetonide, and the antihistamine drugs levocetirizine dihydrochloride, desloratadine, cycloheptadine hydrochloride, have been investigated to confirm the other properties of these drugs. This study aimed to determine the effect of nine known drugs on human salivary α-amylase in vitro and the nature of interactions with structure-activity relationship using molecular docking experiments. The inhibition of human salivary alpha amylase by the six anti-inflammatory and three antihistamine drugs has been carried out using the new method that has been proved in our previous work. Molecular docking has been achieved for the first time for these drugs using the Auto- Dock Vina program. Cyproheptadine hydrochloride presented the highest inhibitory activity against α-amylase with IC50=0.7 mg/ml, while the other drugs showed weak activities (IC50 > 2 mg/ml). We conclude that Cyproheptadine hydrochloride, which was studied by docking experiments, exhibited the best inhibitory activity on salivary α-amylase in vitro & in silico.

New Inhibition Detection Method to Evaluate the Human Salivary Alphaamylase Activity of Some Drugs, Molecular Docking, and SAR Studies.

For the first time, the investigation of six anti-inflammatory drugs and six antihistaminic drugs for inhibitory activities against alpha-amylase has been evaluated using a new inhibition detection method in order to find new treatments for some diseases caused by α-amylase. The first part of this work was devoted to the evaluation of the inhibition activity of these drugs on salivary α-amylase in vitro. Then to study the nature of interactions and structure-activity relationship, using the Autodockvina program for molecular docking. The evaluation of the inhibitory activity of our drugs is achieved using a new method that has proved its sensitivity, quickness, and effectiveness. The results of this study show that betamethasone and loratadine are potent α-amylase inhibitors with IC50 values 0.7mg/ml and 1.03 mg/ml, respectively compared to acarbose with IC50=5.6 μg/ml. The results showed that the loratadine and the betamethasone have a strong potential to inhibit the alpha-amylase.

Variation of human salivary alpha-amylase proteoforms in three stimulation models.

To evaluate the sAA proteoforms' expression during different stimulation situations. This study evaluated the salivary alpha-amylase (sAA) proteoforms' behavior by western blot (WB) analysis and high-resolution mass spectrometry (LC-MS/MS) in different situations that produce increases in sAA activity. For this purpose, six healthy women with a similar body mass index, age, and fit, underwent different sAA stimulation tests, such as acetic acid stimulation, psychological stress using the standardized Trier social stress test, and physical effort using the Cooper treadmill test. The three models showed an increase in sAA activity. The WB demonstrated seven common bands observed in the six women (band one at 59kDa, two at 56kDa, three at 48kDa, four at 45kDa, five at 41kDa, six at 36kDa, and seven at 14kDa), in which sAA protein was identified. The individual WB analysis showed that band two, which corresponded to the native non-glycosylated sAA proteoform, had a higher increase after the three sAA stimulation inducers, and this band was also the only proteoform correlated with sAA activity (r = 0.56, P = 0.001). In addition, when the label-free quantification analysis was performed, the different proteoforms showed different responses depending on the type of stimulation. This preliminary study showed that the diverse sAA proteoforms' expression depends on the different stimulation models. This study opens new perspectives and challenges for the use of the different alpha-amylase proteoforms as possible biomarkers in addition to the sAA activity.

The effectiveness of the RSID confirmatory test kit for human alpha amylase: the effects of environmental factors and substrate materials

The investment in time and money that DNA analyses involves, provides a justification for forensic laboratories to carry out prior tests to ensure the presence of biological fluid in samples. The RSID kit has already been evaluated for forensic use, but its effectiveness on forensic casework samples is almost impossible to predict. Moreover, forensic reports presented before a court of law must justify the methods used to generate their findings. Consequently, trials to know how factors – such as the characteristics of the donor, the substrate and environmental conditions – can affect the kit’s efficacy are essential. This study evaluates some of these factors. For this aim, samples were made using lyophilized human salivary alpha amylase, with known concentrations, on different materials. Then were distributed in two groups, A (not protected) and B (stored and protected) to evaluate possible differences in the effectiveness of the test. The results show how those factors affect the kit’s performance, and, moreover, demonstrate that, contrary to the results that the literature provides for samples in solutions, the kit is more effective on saliva than on alpha amylase. Once again, this highlights the different behaviour of forensic casework samples compared with those that were used in the kit’s evaluation.

Determination of Molar Mass and Its Relationship with Free Energy of Activation: A Case Study on Human Salivary Alpha Amylase

Determination of Molar Mass and Its Relationship with Free Energy of Activation: A Case Study on Human Salivary Alpha Amylase

Anthocyanin composition, antioxidant efficiency, and α-amylase inhibitor activity of different Hungarian sour cherry varieties (Prunus cerasus L.)

Five Hungarian sour cherry cultivars were studied to determine their anthocyanin contents and their possible inhibitory properties. The water and methanol soluble antioxidant capacities were separately assessed by photoluminescence showing values ranged from 3.4μgmg−1 to 15.4μgmg−1, respectively. The “VN1” variety (selected from “Csengődi csokros”) showed the highest antioxidant capacity. The anthocyanin content, measured by pH differential method or isolated by solid phase extraction, was the highest also in “VN1”. Correlation was found between the anthocyanin content and the high antioxidant capacity. The main anthocyanin components were cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside. The presence of malvidin-3,5-O-diglycoside was verified by MALDI-TOF MS. Sour cherry extracts and selected anthocyanins inhibited the human salivary alpha-amylase catalyzed hydrolysis competitively. The lowest IC50 value, 55μgmL−1 or 80μM, was measured for malvidin-3,5-O-diglycoside, for which possible binding modes within the alpha-amylase active site could be investigated in silico using molecular docking and molecular dynamics.

Pouteria ramiflora extract inhibits salivary amylolytic activity and decreases glycemic level in mice

In this study, extracts of plant species from the Cerrado biome were assessed in order to find potential inhibitors of human salivary alpha-amylase. The plants were collected and extracts were obtained from leaves, bark, and roots. We performed a preliminary phytochemical analysis and a screening for salivar alpha-amylase inhibitory activity. Only three botanical families (Sapotaceae, Sapindaceae and Flacourtiaceae) and 16 extracts showed a substantial inhibition (>75%) of alpha-amylase. The ethanolic extracts of Pouteria ramiflora obtained from stem barks and root barks decreased amylolytic activity above 95% at a final concentration of 20 µg/mL. Thus, adult male Swiss mice were treated orally with P. ramiflora in acute toxicity and glycemic control studies. Daily administration with 25, 50 and 100 mg/kg of aqueous extract of P. ramiflora for eight days can reduce significantly body weight and blood glucose level in mice. These data suggest that the crude polar extract of P. ramiflora decreases salivary amylolytic activity while lowering the blood levels of glucose.

Bioelectrode for detection of human salivary amylase

The aim of this study is the development of a novel bioelectrode based on the immobilization of a specific antibody for salivary amylase onto a graphite electrode modified with poly(3-hydroxyphenylacetic) acid. For this purpose, human salivary alpha-amylase was applied to an immunoaffinity-purified anti-alpha-amylase polyclonal antibody. The bioelectrode was incubated with salivary amylase or lysozyme (interfering salivary), and the interaction between the antibody and the enzymes was analyzed through electrochemical impedance spectroscopy. The results indicated the specificity of the bioelectrode to salivary alpha-amylase. Therefore, the combination of the graphite electrode with poly(3-hydroxyphenylacetic acid) appears to be a promising strategy for antigen immobilization and other biological recognition elements, thus presenting the potential for the production of a label-free biochip.

Laryngoscopy and endotracheal intubation induced changes in human salivary alpha amylase activity - Associations with adrenergic activity

Laryngoscopy and endotracheal intubation induced changes in human salivary alpha amylase activity - Associations with adrenergic activity

Evaluation of Different Teas against Starch Digestibility by Mammalian Glycosidases

Current work investigated the ability of different tea (green, oolong and black teas) in inhibiting human salivary alpha-amylase (HSA) and mammalian alpha-glucosidase (AGH). The inhibitory profiles were correlated to their major polyphenol content (theaflavins and catechins). The fully fermented black tea was demonstrated to be most potent in inhibiting HSA and AGH (IC50 of 0.42 to 0.67 and 0.56 to 0.58 mg of tea leaves/mL respectively). Its capability in retarding the digestion of a real food system (rice noodle) was further elucidated with an in vitro digestion study. Results indicated that black tea was able to retard starch digestion moderately, thereby allowing a gradual reduction of sugar liberation. Polyphenolic profile analysis suggested that the oxidized catechins, theaflavins, may be responsible for its activity. We have found that refractive index (RI) measurement is a rapid, direct, and highly convenient method for quantifying the degree of enzymatic starch digestion and kinetics. The RI method has good linearity range, limit of detection (0.1596 mg/mL, maltose equivalent) and limit of quantitation (0.6312 mg/mL) and was successfully applied in our study.

Contribution of AmyA, an extracellular α‐glucan degrading enzyme, to group A streptococcal host–pathogen interaction

alpha-Glucans such as starch and glycogen are abundant in the human oropharynx, the main site of group A Streptococcus (GAS) infection. However, the role in pathogenesis of GAS extracellular alpha-glucan binding and degrading enzymes is unknown. The serotype M1 GAS genome encodes two extracellular proteins putatively involved in alpha-glucan binding and degradation; pulA encodes a cell wall anchored pullulanase and amyA encodes a freely secreted putative cyclomaltodextrin alpha-glucanotransferase. Genetic inactivation of amyA, but not pulA, abolished GAS alpha-glucan degradation. The DeltaamyA strain had a slower rate of translocation across human pharyngeal epithelial cells. Consistent with this finding, the DeltaamyA strain was less virulent following mouse mucosal challenge. Recombinant AmyA degraded alpha-glucans into beta-cyclomaltodextrins that reduced pharyngeal cell transepithelial resistance, providing a physiologic explanation for the observed transepithelial migration phenotype. Higher amyA transcript levels were present in serotype M1 GAS strains causing invasive infection compared with strains causing pharyngitis. GAS proliferation in a defined alpha-glucan-containing medium was dependent on the presence of human salivary alpha-amylase. These data delineate the molecular mechanisms by which alpha-glucan degradation contributes to GAS host-pathogen interaction, including how GAS uses human salivary alpha-amylase for its own metabolic benefit.

Molecular characterization of group A Streptococcus maltodextrin catabolism and its role in pharyngitis

We previously demonstrated that the cell-surface lipoprotein MalE contributes to GAS maltose/maltodextrin utilization, but MalE inactivation does not completely abrogate GAS catabolism of maltose or maltotriose. Using a genome-wide approach, we identified the GAS phosphotransferase system (PTS) responsible for non-MalE maltose/maltotriose transport. This PTS is encoded by an open reading frame (M5005_spy1692) previously annotated as ptsG based on homology with the glucose PTS in Bacillus subtilis. Genetic inactivation of M5005_spy1692 significantly reduced transport rates of radiolabelled maltose and maltotriose, but not glucose, leading us to propose its reannotation as malT for maltose transporter. The DeltamalT, DeltamalE and DeltamalE:malT strains were significantly attenuated in their growth in human saliva and in their ability to catabolize alpha-glucans digested by purified human salivary alpha-amylase. Compared with wild-type, the three isogenic mutant strains were significantly impaired in their ability to colonize the mouse oropharynx. Finally, we discovered that the transcript levels of maltodextrin utilization genes are regulated by competitive binding of the maltose repressor MalR and catabolite control protein A. These data provide novel insights into regulation of the GAS maltodextrin genes and their role in GAS host-pathogen interaction, thereby increasing the understanding of links between nutrient acquisition and virulence in common human pathogens.

Flavonoids for Controlling Starch Digestion: Structural Requirements for Inhibiting Human α-Amylase

In this study we investigated the structural requirements for inhibition of human salivary alpha-amylase by flavonoids. Four flavonols and three flavones, out of the 19 flavonoids tested, exhibited IC50 values less than 100 microM against human salivary alpha-amylase activity. Structure-activity relationships of these inhibitors by computational ligand docking showed that the inhibitory activity of flavonols and flavones depends on (i) hydrogen bonds between the hydroxyl groups of the polyphenol ligands and the catalytic residues of the binding site and (ii) formation of a conjugated pi-system that stabilizes the interaction with the active site. Our findings show that certain naturally occurring flavonoids act as inhibitors of human alpha-amylase, which makes them promising candidates for controlling the digestion of starch and postprandial glycemia.

Bifunctional inhibitor of α-amylase/trypsin from wheat grain

A trypsin inhibitor, isolated from whole-wheat grain (Triticum aestivum L.) by the method of bio-specific chromatography on trypsin-Sepharose, was potent in inhibiting human salivary alpha-amylase. The bi-functional alpha-amylase/trypsin inhibitor was characterized by a narrow specificity for other alpha-amylases and proteinases. The high thermostability of the inhibitor was lost in the presence of SH group-reducing agents. The inhibitor-trypsin complex retained its activity against alpha-amylase. The inhibitor-alpha-amylase complex was active against trypsin. Studies of the enzyme kinetics demonstrated that the inhibition of alpha-amylase and trypsin was noncompetitive. Our results suggest the existence of two independent active sites responsible for the interaction with the enzymes.