Enzyme Inhibition Method Research Articles

Inquiry lipaseoring the mechanism of pancreatic lipase inhibition by isovitexin based on multispectral method and enzyme inhibition assay.

Isovitexin is a main natural flavonoid component in various plants. Currently, the inhibitory effect of isovitexin on pancreatic lipase (PL) and its mechanism have not been elucidated yet. In the present study, we investigated the inhibitory effect of isovitexin on PL, as well as its interaction mechanism, using enzyme inhibition methods, spectroscopic analysis, and molecular simulations. Results showed that isovitexin possessed significant PL inhibitory activity, with IC50 values of 0.26 ± 0.02 mM. The interaction between isovitexin and PL was dominated by static quenching, and mainly through hydrogen bonding and hydrophobic interaction forces. Analysis of fluorescence spectroscopy confirmed that isovitexin binding altered the conformation of the PL. Circular dichroism (CD) spectrum indicated that isovitexin altered the secondary structure of PL by decreasing the α-helix content and increasing the β-fold content. Molecular simulations further characterize the conformational changes produced by the interaction between isovitexin with PL. The performed study may provide a new insight into the inhibitory mechanism of isovitexin as a novel PL inhibitor.

Highly Sensitive and Rapid Screening Technique for the Detection of Organophosphate Pesticides and Copper Compounds Using Bifunctional Recombinant TrxA-PvCarE1.

Enabling the detection of organophosphate pesticide (OP) residues through enzyme inhibition-based technology is crucial for ensuring food safety and human health. However, the use of acetylcholinesterase, the primary target enzyme for OPs, isolated from animals in practical production poses challenges in terms of sensitivity and batch stability. To address this issue, we identified a highly sensitive and reproducible biorecognition element, TrxA-PvCarE1, derived from red kidney beans and successfully overexpressed it in Escherichia coli. The resulting recombinant TrxA-PvCarE1 exhibited remarkable sensitivity toward 10 OPs, surpassing that of commercial acetylcholinesterase. Additionally, this approach demonstrated the capability to simultaneously detect copper compounds with high sensitivity, expanding the range of pesticides detectable using the traditional enzyme inhibition method. Spiking recovery tests conducted on cowpea and carrot samples verified the suitability of the TrxA-PvCarE1-based technique for real-life sample analysis. In summary, this study highlights a promising comprehensive candidate for the rapid detection of pesticide residues.

Antidiabetic and Antibacterial Activities of Syzygium cumini Seeds

Bioactive compounds of plants have been used in treating various diseases from ancient times. In this study, Syzygium cumini seed extract was studied for its antioxidant, antibacterial, and anti-diabetic potentials. The antioxidant potential was evaluated by DPPH assay. Whereas, the antibacterial feature was tested against Staphylococcus aureus, Escherichia coli, Salmonella typhi, and Klebsiella pneumonia by agar well diffusion method. The in vitro anti-diabetic potential was performed by the α-amylase enzyme inhibition method using CNPG3 (2-chloro-4-nitrophenyl-α-D-maltotriose) as a substrate. The methanolic extract showed a total phenolic content of 128.8 ± 1.44 mg GAE/g and a total flavonoid content of 115.6 ± 8.01 mg QE/g. The extract scavenged free radicals where strong antioxidant activity was observed with IC50 values of 91.63 ± 6.54 μg/mL in the concentration range of 50-6.25 μg/mL. The DCM and EA fractions exhibited moderate antidiabetic potentials with IC50 values of 92.1 ± 0.5 μg/mL, 150.8 ± 0.8 μg/mL, and 42.2 ± 4.6 μg/mL in the concentration range of 400-25 μg/mL against acarbose standard having IC50 values of 6.02 ± 0.1 μg/mL in the concentration range 50-1.6 μg/mL. The methanolic extract having ZOI (19mm) showed antibacterial activity however standard neomycin demonstrated ZOI (22.5 mm) against S. aureus at the dosage of 50 μg/mL. Methanolic extract having ZOI (20 mm) demonstrated effective antibacterial activity when compared to standard neomycin with ZOI (15mm) against K. pneumonia at the dosage of 50 μg/mL.

Carboxylesterase from edible legume seeds and their sensitivity to organophosphate and carbamate pesticides

Carboxylesterase from edible legume seeds, including black gram bean (Vigna mungo (L.) Hepper), mung bean (Vigna radiata (L.) Wilczek) and red bean (Vigna umbellata (Thunb.) Ohwi and Ohashi) has been partially purified using the ammonium sulphate salting-out procedure. The specific activity of the partially purified carboxylesterase extracted from black gram bean, mung bean and red bean are 3.96±0.26, 1.51±0.10 and 1.29±0.08 U/mg, respectively (p<0.05). The carboxylesterase from black gram bean showed very high sensitivity to 24 types of organophosphate pesticides (OPs) and 7 types of carbamate pesticides (CAs) at the concentrations of 0.001-3.0 µg/ml. The limit of detection (LOD) obtained for OPs and CAs pesticide residues were 0.002 and 0.01 mg/kg, respectively. For performance assessment of methods for pesticide determination in vegetables, all performance parameter values, including sensitivity, accuracy, and specificity of the carboxylesterase enzyme inhibition method, are 100% compared with commercial pesticide test kits and are 70.5%, 90%, and 100%, respectively, compared with chromatographic method analysis.

Phytochemical Composition, In Vitro Antimicrobial, Antioxidant, and Enzyme Inhibition Activities, and In Silico Molecular Docking and Dynamics Simulations of Centaurea lycaonica: A Computational and Experimental Approach.

Centaurea lycaonica is a local endemic species from the Centaurea L. genus. The Centaurea species has a wide range of usage in treating diseases in folk medicine. There are limited biological activity studies on this species in the literature. This study investigated enzyme inhibition and antimicrobial activity, antioxidant effect, and chemical content of extract and fractions of C. lycaonica. Enzyme inhibition activity was tested by α-amylase, α-glucosidase, and tyrosinase enzyme inhibition methods and antimicrobial activity by the microdilution method. The antioxidant activity was investigated using DPPH•, ABTS•+, and FRAP tests. The chemical content was determined by LC-MS/MS. The methanol extract showed the highest activity for α-glucosidase and α-amylase, even surpassing the positive control acarbose, with IC50 values of 56.333 ± 0.986 and 172.800 ± 0.816 μg/mL, respectively. Additionally, the ethyl acetate fraction also exhibited high activity for α-amylase with an IC50 value of 204.067 ± 1.739 μg/mL and tyrosinase with an IC50 value of 213.900 ± 1.553 μg/mL. Moreover, this extract and fraction were found to have the highest total phenolic and flavonoid contents and antioxidant activity. Additionally, LC-MS/MS analyses of active extract and fraction revealed mainly the presence of phenolic compounds and flavonoids. In silico molecular docking and molecular dynamics simulation studies of determining compounds apigenin and myristoleic acid, common in CLM and CLE extracts and active against α-glucosidase and α-amylase, were performed. In conclusion, methanol extract and ethyl acetate fraction showed potential enzyme inhibition and antioxidant activity as a natural agent. Molecular modeling studies corroborate the findings of in vitro activity analyses.

In silico and biological activity evaluation of quercetin-boron hybrid compounds, anti-quorum sensing effect as alternative potential against microbial resistance

Boronic acid compounds and the natural flavonoid compound quercetin were handled to synthesize two novel ligands encoded as B1(2,2′-(1,4-phenylenebis (benzo [1,3,2] dioxaborole-2,5-diyl)) bis (3,5,7-trihydroxy-4H- chromen-4-one) and B2(3.3.6. 3,5,7-trihydroxy-2-(2-(6-methoxypyridin-3-yl)benzo[d][1,3,2]dioxaborol-5-yl)− 4 H-chromene-4). Antioxidant activities of ligands were investigated by DPPH, ABTS and CUPRAC methods. Cholinesterase inhibition effects of ligands were determined by acetylcholinesterase and butyrylcholinesterase enzyme inhibition methods, cytotoxic effects of ligands were applied to healthy breast and colon cancer cell lines by MTT method, and urease and tyrosinase enzyme activities were determined. Antimicrobial properties of the compounds were analyzed by detecting their anti-QS potentials on Chromobacterium violaceum biosensor strain. Both compounds were found to have significant antioxidant effects compared to controls. It was determined that the compound B1 at 1–10 µg/mL was more active than the reference compounds (α-TOC and BHT). Moreover, the enzyme activity studies on ligands demonstrated that acetylchoinesterase and butyrylcholinesterase enzyme inhibitions were higher than the reference compounds. As expected, boron derivatives exhibited respectable activity against the biofilms of Escherichia coli (E. coli) and P. aeruginosa (P. aeruginosa). These results demonstrate the potential applicability of boron derivatives in the treatment of biofilm-associated infections and provide a practical strategy for the design of new boron-based antimicrobial materials. In silico molecular docking studies were performed on ligands to identify newly synthesized compounds. The binding parameter values and binding sites of the compounds were also determined. In conclusion, our studies showed that newly synthesized hybrid compounds could be solutions for antimicrobial resistance and enzyme-related disorders.

In Vitro Antidiabetic Activity Of Apis dorsata Binghami Nest Extract

Indonesia is ranked 7th out of 10 countries with the highest number of people with diabetes mellitus, which is estimated to increase every year.α -amylase is an enzyme that catalyzes the hydrolysis of α-1,4 glycosidic bonds of polysaccharides to produce dextrins, oligosaccharides, maltose and D-glucose. Apis dorsata Binghami honeycomb contains secondary metabolites that have antidiabetic potential, including inhibiting the action of the α-amylase enzyme. The purpose of this study was to determine the in vitro antidiabetic potential of Apis dorsata binghami nest extract. This study uses a descriptive research method where the research data are obtained through laboratory experiments. Honeycomb extraction using maceration method. Analysis of total flavonoid content using UV-Vis spectrophotometric method. Antidiabetic potency test using -amylase enzyme inhibition method. The results of the extraction study showed that the % yield of the extract was 11.82% with a brownish yellow color. Analysis of the total flavonoid content showed the results of 3.33 mgQE/g. The inhibitory activity of the -amylase enzyme, the IC50 value of the extract obtained was 158.48±7.42 g/mL, the acarbose value was 165.96±7.08 g/mL. These results indicate that the extract of A.dorsata Binghami nest has hyperglycemic activity by inhibiting complex carbohydrate hydrolyzing enzymes such as inhibition of the -amylase enzyme which is better than acarbose because the IC50 value of the extract is smaller. If the IC50 value is smaller then the enzyme inhibition is stronger. For further research, it is recommended to use a more specific enzyme, namely the -glucosidase enzyme.

Evaluation of the Chemical Composition, Antioxidant and Antidiabetic Activity of Rhaponticoides iconiensis Flowers: Effects on Key Enzymes Linked to Type 2 Diabetes In Vitro, In Silico and on Alloxan-Induced Diabetic Rats In Vivo.

Diabetes mellitus (DM) is one of the globally worst killer diseases. In this study, the in vitro and in vivo antidiabetic activity and antioxidant capacity were determined and the phytochemical analyses were carried out on flower extract and sub-extracts of Rhaponticoides iconiensis. The in vitro antidiabetic activity was tested with α-amylase and α-glucosidase enzyme inhibition methods and an in vivo OGTT test in healthy and alloxan-induced rats. Although, the antioxidant activity was investigated with DPPH●, ABTS●+ and FRAP tests, the phytochemical composition analysis was carried out by LC-MS/MS. The highest α-glucosidase and α-amylase activity even from positive control acarbose were found in the ethyl acetate sub-extract of R. iconiensis (IC50 = 11.737 ± 0.823 µg/mL and 84.247 ± 0.721 µg/mL, respectively). This sub-extract also was active according to the results of in vivo tests. Moreover, the highest antioxidant activity on DPPH● (IC50 = 0.126 ± 0.002 mg/mL), FRAP (at a concentration of 1 mg/mL equivalent to 3112.052 ± 2.023 mmol Fe2+) and ABTS+● (at a concentration of 0.5 mg/mL equivalent to 0.608 ± 0.005 µM Trolox) tests. In addition, LC-MS/MS analyses of the active sub-extract revealed mainly the presence of patuletin, patuletin 3,7-diglucoside, naringin and 3,4-dicaffeoylquinic acid detected in the active sub-extract. In silico molecular docking and dynamics simulations studies were performed on these compounds with α-amylase and α-glucosidase enzymes for protein-ligand interactions and stability.

Chemical Composition, in vitro Antioxidant, and Antidiabetic Potential of Essential Oil from Flowers of Rhaponticoides iconiensis

The present study describes in vitro antidiabetic activity, antioxidant capacity and phytochemical analyses of essential oil from flower of Rhaponticoides iconiensis (Hub.-Mor.) M.V.Agab. & Greuter. In this study, the antidiabetic activity of essential oil was carried out by α-amylase and α-glucosidase enzyme inhibition methods, the antioxidant activity was investigated with DPPH●, ABTS●+ and FRAP tests and the phytochemical composition analysis was carried out by GC/MS and GC-FID. According to the results, the highest α-amylase and ABTS+● activity even from positive control were found in essential oil (IC50=154.33±3.88 µg/mL and at a concentration of 0.25 mg/mL equivalent to 0.607±0.95 µM Trolox, respectively). Moreover, the highest antioxidant activity on FRAP (at a concentration of 1 mg/mL equivalent to 508.50±1.057 mmol Fe2+) and moderate effect on DPPH● (33.10%) tests were observed. Besides that, the results of GC/MS analyses of essential oil revealed the presence of 1-undecene, γ-eudesmol and β-caryophyllene as a major compounds. In conclusion, the essential oil from this species has potential antidiabetic and antioxidant activity and these activities may be investigated in detail in the future.

Benzothiazole sulfonate derivatives bearing azomethine: Synthesis, characterization, enzyme inhibition, and molecular docking study

Enzyme inhibition is one of the most applied ways to find new diagnostic facilities for the prevention and treatment of many health problems. Tyrosinase and pancreatic lipase inhibitions have been used for the treatment of skin pigmentation problems and obesity, respectively. This study mainly focused to evaluate the enzyme inhibitions of the newly synthesized sulfonate derivatives (3a-3i) for tyrosinase and pancreatic lipase. The structural characterizations of the 2-(((5-methylbenzo[d]thiazol-2-yl)imino)methyl)phenyl sulfonate derivatives were performed by 1H NMR, 13C NMR, and HR-MS analyses. According to the in vitro enzyme inhibition methods, compound 3e had the highest tyrosinase inhibitory activity (46.9 ± 3.6 µM IC50 value). On the other hand, the effective pancreatic lipase inhibitory activities of the compounds 3f, 3e, and 3g were determined from their low IC50 values 58.3 ± 6.7 µM, 59.5 ± 13.8 µM, and 64.8 ± 6.3 µM, respectively. Molecular docking studies were also conducted for the synthesized compounds with tyrosinase and pancreatic lipase enzymes separately. Finally, the ADME studies were carried out to determine the pharmacokinetics, drug similarities, and medicinal properties of the target compounds..

Chemical Changes in the Broccoli Volatilome Depending on the Tissue Treatment.

The storage of plant samples as well as sample preparation for extraction have a significant impact on the profile of metabolites, however, these factors are often overlooked during experiments on vegetables or fruit. It was hypothesized that parameters such as sample storage (freezing) and sample pre-treatment methods, including the comminution technique or applied enzyme inhibition methods, could significantly influence the extracted volatile metabolome. Significant changes were observed in the volatile profile of broccoli florets frozen in liquid nitrogen at −20 °C. Those differences were mostly related to the concentration of nitriles and aldehydes. Confocal microscopy indicated some tissue deterioration in the case of slow freezing (−20 °C), whereas the structure of tissue, frozen in liquid nitrogen, was practically intact. Myrosinase activity assay proved that the enzyme remains active after freezing. No pH deviation was noted after sample storage - this parameter did not influence the activity of enzymes. Tissue fragmentation and enzyme-inhibition techniques applied prior to the extraction influenced both the qualitative and quantitative composition of the volatile metabolome of broccoli.

In vitro study of Cratoxylum glaucumStem ethyl acetate extract as antidiabetic

Diabetes mellitus (DM) is a disease characterized by hyperglycemia due to decreased insulin secretion, or decreased insulin sensitivity, or both. If insulin is not available or the amount is low, then glucose cannot enter the cells and will continue to be in the bloodstream. DM can cause chronic complications such as eye complications, skin infections and stroke. DM treatment takes a long time by using synthetic drugs that will cause side effects, therefore the search for herbal-based drugs is the community’s choice. Idat plant (Cratoxylum glaucum) is a local plant from Bangka Belitung. Several studies on the genus Cratoxylum from China and Thailand showed a very high inhibitory ability against the inhibition of a-glucosidase enzymes, protein tyrosine phosphate, and carbohydrate hydrolysis enzymes because they contain phenolic compounds anthraquinones and xanthones. Therefore, the importance of more intensive research on C. glaucum species regarding the content of secondary metabolites in C. glaucum stems and their antidiabetic power. The extraction method in this research is maceration with ethyl acetate solvent. Qualitative examination of phenolic and flavonoid content using reagents, as well as antidiabetic testing was carried out in vitro with dengan α-glucosidase enzyme inhibition method. Based on qualitative testing of phenolic and flavonoid compounds, the content of these compounds was obtained. antidiabetic test showed that the-glucosidase inhibitor of ethyl acetate extract had very strong activity with an IC50 of 4.21 g/mL. Therefore, the stem of the idat plant can be used as a therapy for the treatment of diabetes mellitus.

Single crystal, Hirshfeld surface, DFT analyses of (E)‐2‐(2‐chloro‐6‐fluorobenzylidene)hydrazinecarbothioamide: Elastase inhibition and DNA binding studies

AbstractHere, a new (E)‐2‐(2‐chloro‐6‐fluorobenzylidene)hydrazinecarbothioamide hybrid 3 is reported for synthesis by using simple protocol. Compound was characterized, and its crystal structure was resolved by single crystal X‐ray analysis. The molecular structure showed perfect planarity with the 2,6‐halogen atoms disordered over the two positions. Hirshfeld surface (HS) analysis indicated H‐bonding and van der Waals interactions as leading intermolecular interactions in the crystal structure with most important contributions as H … H (24.10%), H … S/S … H (20.80%) and H … Cl/Cl … H (13.50%). Density functional theory (DFT) analysis further highlighted on molecular orbital's energy transitions and comparison of absorption maxima with experimentally calculated λmax. Crystal was evaluated against elastase enzyme by using molecular docking and in vitro enzyme inhibition methods. An intuitive look at elastase inhibition demonstrated higher binding efficacy and lower IC50. Results from both studies have shown potent inhibitory activity of 3 against elastase. Kb and ΔG for 3‐DNA complex evaluated from molecular docking and UV/visible spectroscopy efficiently complemented each other, and the binding mode observed through docked structure was verified by viscometry. Theoretical and experimental investigations have provided evidence of substantial binding of the title compound with the DNA via intercalation.

Enhanced Secretory Expression and Surface Display Level of Bombyx mori Acetylcholinesterase 2 by Pichia pastoris Based on Codon Optimization Strategy for Pesticides Setection.

The cholinesterase-based spectrophotometric assay, also called enzyme inhibition method, is a good choice for rapid detection of organophosphate pesticides (OPs) and carbamate pesticides (CPs). Obviously, the cholinesterase is the core reagent in enzyme inhibition method. In our previous work, a recombinant acetylcholinesterase 2 from Bombyx mori (rBmAChE2) was expressed in yeast successfully and exhibited great sensitivity. However, the yield of rBmAChE2 is not desirable. In this study, a codon optimization strategy was employed to enhance the yield of rBmAChE2 in Pichia pastoris GS115. Results showed that by replacing 6 key rare codons and increasing the percentage of bases G and C up to 46.85%, codon adaptation index (CAI) of Bombyx mori acetylcholinesterase 2 (bmace2) gene was improved from 0.70 to 0.81. After being transformed into Pichia pastoris GS115 via electroporation, the expression transformant can produce 139.7 U/mL secretory codon-optimized rBmAChE2 (opt-rBmAChE2) in the culture supernatant, 3.62 times higher than that of strain bearing the wild-type bmace2 gene. Meanwhile, opt-rBmAChE2 displayed on the yeast surface was up to 2280.02 U/g, 2.8 times higher than wild-type displayed rBmAChE2. In addition, either secretory or surface-displayed opt-rBmAChE2 maintained the similar sensitivities to the wild-type rBmAChE2 for tested inhibitors. Furthermore, the detection limits of the opt-rBmAChE2-based enzyme inhibition method for 10 kinds of OPs or CPs (0.01-2.69 mg/kg) were lower than most of the indexes present in current standard method (GB/T 5009.199-2003) or the maximum residue limits (GB 2763-2019) in China. The results might contribute to the utilization of rBmAChE2 for pesticide residue screening detection in practice.

3,6-dimethyl ester-α-mangostin Compound Modified from Isolate α-mangostin Garcinia Mangostana Linn

Isolate α-mangostin (1) is a phenolic compound derived from oxygenated and prenylated xanthones and major compounds obtained from the fruit peel of G. mangostana Linn., Which is stated to have inhibitory activity against α-glucosidase enzymes, serves to determine the antidiabetic activity resulting in IC50 value of 29,92 µM, is of the nature moderate to positive control (acarbose) with an IC50 value of 4.55 µM. A modified compound of α-mangostin (1) with acetic anhydride obtained by 3,6-di-methyl ester-α-mangostin (2) derivative showed the inhibitory value of α-glucosidase (IC50 13,89 μM), this value is better than the activity inhibition of α-mangostin (1), but not as active as the positive control value of the acarbose compound. The separation process to obtain α-mangostin isolates from the fruit peel of G. mangostana Linn was obtained by maceration method with ethyl acetate solvent, followed by refraction using a vacuum liquid chromatography (KCV) method over silica gel (Merck 60 G) and eluted using eluent (n-hexane: ethyl acetate) with increasing polarity, to produce as much pure crystal (21.66 g), yield (24%). While the structural characterization of the two compounds was carried out using UV-Vis, IR, HRESIMS, 1H-NMR and 13C-NMR spectroscopic methods, the antidiabetic testing was carried out using the α-glucosidase enzyme inhibition method.

Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides

Acetylcholinesterase (AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method. An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by Pichia pastoris. The pPIC9K-Drosophilamelanogaster acetylcholinesterase (DmAChE)-like expression vector was constructed by codon optimization of this mutant strain, which was transformed into P. pastoris GS115, and positive clones were selected on yeast peptone dextrose (YPD) plate with G418 at 4.0 mg/mL. The GS115-pPIC9K-DmAChE-like strain was subjected to 0.5% methanol induction expression for 120 h, with a protein band at 4.3 kDa found by the tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of the fermentation supernatant. After preliminary purification by ammonium sulfate precipitation, the enzyme activity was detected to be 76.9 U/(mL⋅min). In addition, the pesticide sensitivity test proved that DmAChE-like is selective and sensitive to organophosphorus pesticides.

Double-enzymes-mediated Fe2+/Fe3+ conversion as magnetic relaxation switch for pesticide residues sensing

It is a great challenge to develop a newly rapid and accurate detection method for pesticide residues. In this work, based on acetylcholinesterase (AChE) and choline oxidase (CHO), a double-enzymes-mediated Fe2+/Fe3+ conversion as magnetic relaxation switch was explored for the measurement of acetamiprid residue. In the double-enzymes reactions, acetylcholine chloride (ACh) can be catalyzed to produce choline by AChE, which is successively hydrolyzed to betaine and hydrogen peroxide (H2O2) by CHO. According to the enzyme inhibition principle, AChE activity will be inactivated in the presence of acetamiprid, thus leading to the less production of H2O2. Wherein, Fe2+, ACh, AChE and CHO were optimized as the reaction substrates. In the reaction system, acetamiprid can be reflected by the transverse relaxation time (T2) that related with H2O2 mediated Fe2+ variations, which was further developed as an enzyme cascade amplification method. The detection linear range is 0.01∼1000 μg mL−1 (R2 = 0.99), and the limit of detection (LOD) is 2.66 ng mL−1 (S/N = 3, n = 3), behaving a 335-fold improvement in LOD than that of traditional enzyme inhibition method (0.89 μg mL−1). This method can realize “one-step mixing” detection of acetamiprid, which makes it a promising analytical tool for monitoring pesticide residue in complicated samples.

An Overview on the Mechanisms and Applications of Enzyme Inhibition-Based Methods for Determination of Organophosphate and Carbamate Pesticides

Acetylcholinesterase inactivating compounds, such as organophosphate (OP) and carbamate (CM) pesticides, are widely used in agriculture to ensure sustainable production of food and feed. As a consequence of their applications, they would result in neurotoxicity, even death. In this essence, the development of enzyme inhibition methods still shows great significance as rapid detection techniques for on-site large-scale screening of OPs and CMs. Initially, mechanisms and applications of various enzyme-inhibition-based methods and devices, including optical colorimetric assay, fluorometric assays, electrochemical biosensors, rapid test card, and microfluidic device, are highlighted in the present overview. Further, to enhance the enzyme sensitivity for detection; alternative enzyme sources or high yield enrichment methods (such as abzyme, artificial enzyme, and recombinant enzyme), as well as enzyme reactivation and identification, are also addressed in this comprehensive overview.

Extraction and characterization of phycocyanin from Spirulina platensis and evaluation of its anticancer, antidiabetic and antiinflammatory effect

The phycocyanin was purified by Sephadex- G-100 and RP-HPLC and protein content was found to be 52.82% and the high purity fraction was collected and RP-HPLC analysis of fractionated phycocyanin, the α-subunit and β-subunit were detected in 4.9 and 11.1(mAU). The frequency of peak 1456.26 cm−1 has showed the CH2 bending vibration and the protein amide II band was detected at 1539.20 cm−1 (CO stretching) and 2358.94 cm−1. In 1H NMR analysis, 14 chemical shifts (δ) were observed and signals confirmed namely alkyl halide, alkene, aldehyde proton and carboxylic acid. The in vivo anticancer effect was assessed by MTT assay against HepG-2 cell lines and in vivo antidiabetic effect was carried out through α-amylase and β-glucosidase enzyme inhibition methods. The promising anticancer effect 68% was noticed at the concentration of 500 μg/ml and lower anticancer effect was noticed at the concentration of 100 μg/ml against Hep-G2 cell lines. The α-amylase and β-glucosidase enzyme inhibition of phycocyanin showed dose dependent and maximum inhibition effect at 250 μg/ml. Phycocyanin anti-inflammatory effect such as inhibition of albumin denaturation, antiproteinase, hypotonicity-induced haemolysis and anti-lipoxygenase activities have been recorded maximum level at 500 μg/ml. Phycocyanin have complex structure and high molecular weight with more biomedical applications for drug development.

The In Vitro Anti-Diabetic Activity of Lime Peels (Citrus amblycarpa (Hassk.) Ochse)

There are various potential natural anti-diabetic drugs; one of them is lime peel or Citrus amblycarpa. This study was aimed to explore the anti-diabetic activity and phytochemical content of lime peels. This study was an experimental study that used the post-test only control group design. The lime peels that were collected from the Berastagi fruit market in Medan, North Sumatera were extracted using 70% ethanol by maceration methods. The phytochemical screening identified the presence of phenolic, steroid/triterpenoid, terpenoid, saponin, flavonoid, tannin, and alkaloid. Meanwhile, the anti-diabetic activity of lime peels was evaluate using the α-glucosidase enzyme that was gotten from Saccharomyces cerevisiae by α-glucosidase enzyme inhibition methods. Percent of inhibition was express as Mean ± SD and analyzed by One Way ANOVA, Tukey HSD Post Hoc Test, and followed by linear regression. The result of this study showed that there is a significant difference in percentage inhibition α-glucosidase enzyme in each concentration, and it had an IC50 Value amount of 125.93 ± 9.14 µg/mL. The phytochemical content of the lime peels was flavonoid, phenol, steroid/triterpenoid, and alkaloid. Hence, the lime peel has anti-diabetic activity by inhibition of the α-glucosidase enzyme.