New Specific α-Glucosidase Inhibitor Flavonoid from Thymelaea tartonraira Leaves: Structure Elucidation, Biological and Molecular Docking Studies.

The phytochemical investigation of Thymelaea tartonraira leaves led to the isolation and characterization of six compounds, including one new flavonoid glycoside identified as hypolaetin 8-O-ß-D-galactopyranoside (4) along with five known compounds, daphnoretin (1), triumbelletin (2), genkwanin (3), tiliroside (5) and yuankanin (6). Their structures were established based on spectroscopic methods, such as UV, IR, NMR, and HR-ESI-MS. Triumbelletin (2) and tiliroside (5) were isolated for the first time from T. tartonraira leaves. The antioxidant property of all isolated compounds was tested based on DPPH, FRAP and total antioxidant capacity assays. Compound 4 displayed an antioxidant potency more interesting than vitamin C with an IC50 =15.00±0.50 µg/ml, followed by compound 5. Furthermore, the both compounds 4 and 5 were tested for their α-amylase inhibitory activity in-vitro. Compound 4 displayed higher potency to inhibit α-amylase, with an IC50 =46.49±2.32 µg/ml, than compound 5, with an IC50 =184.2±9.2 µg/ml, while the reference compound acarbose presented the highest potency to inhibit α-amylase with an IC50 =0.44±0.022 µg/ml. Compound 4 displayed a strong inhibitory ability of α-glucosidase activity approximately twice more than the reference compound, acarbose, with IC50 values of 60.00±3.00 and 125.00±6.25 µg/ml, respectively. Thus, compound 4 exhibited a specific inhibitory activity for α-glucosidase. The molecular docking studies have supported our findings and suggested that compound 4 has been involved in various binding interactions within the active site of both enzymes α-amylase and α-glucosidase.

Isolation of Chalcomoracin as a Potential α-Glycosidase Inhibitor from Mulberry Leaves and Its Binding Mechanism.

Diabetes is a chronic metabolic disease, whereas α-glucosidases are key enzymes involved in the metabolism of starch and glycogen. There is a long history of the use of mulberry leaf (the leaf of Morus alba) as an antidiabetic herb in China, and we found that chalcomoracin, one of the specific Diels-Alder adducts in mulberry leaf, had prominent α-glucosidase inhibitory activity and has the potential to be a substitute for current hypoglycemic drugs such as acarbose, which have severe gastrointestinal side effects. In this study, chalcomoracin was effectively isolated from mulberry leaves, and its α-glucosidase inhibition was studied via enzymatic kinetics, isothermal titration (ITC) and molecular docking. The results showed that chalcomoracin inhibited α-glucosidase through both competitive and non-competitive manners, and its inhibitory activity was stronger than that of 1-doxymycin (1-DNJ) but slightly weaker than that of acarbose. ITC analysis revealed that the combination of chalcomoracin and α-glucosidase was an entropy-driven spontaneous reaction, and the molecular docking results also verified this conclusion. During the binding process, chalcomoracin went into the "pocket" of α-glucosidase via hydrophobic interactions, and it is linked with residues Val544, Asp95, Ala93, Gly119, Arg275 and Pro287 by hydrogen bonds. This study provided a potential compound for the prevention and treatment of diabetes and a theoretical basis for the discovery of novel candidates for α-glycosidase inhibitors.

Phytochemical Composition, Antibacterial, Antioxidant and Antidiabetic Potentials of Cydonia oblonga Bark.

Cydonia oblonga is a medicinal plant that is used to treat a number of health complications in traditional medication systems. The objective of this study was to evaluate the phytochemical composition, and antibacterial, antioxidant, and ant-diabetic potentials of methanolic extracts of Cydonia oblonga bark. The Cydonia oblonga bark extraction was fractionated through HPLC and seven purified fractions labeled as F1, F2, F3, F4, F5, F6, and F7 were obtained. The HPLC-UV analysis of methanolic extract showed the presence of a number of possible compounds. The GC-MS and HPLC analysis confirmed the presence of the following bioactive compounds in the crude extract and purified fractions: malic acid, mandelic acid, quercetin, caffeic acid, catechin hydrate, as morin (HPLC analysis), BIS-(2-ethylhexyl)phthalate and diisooctyl phthalate (F1), carbamide (F2, used as fertilizer), octasiloxane and dimethylsiloxanecyclictrimer (F3), silicic acid and cyclotrisiloxane (F4), 6-AH-cAMP, 4H-cyclopropa[5',6']benz[1',2',7,8]azule, and 4-(4-chlorophenyl)-3-morpholinepyrol-2-yl)-butenedioic acid (F5), isopropyamine (F6), and 1-propylhydrazine (F7). The extract and purified fractions were then tested for biological activities. All the purified fractions and methanolic extract showed effective antibacterial activity; however, the highest activity was recorded for methanolic extract against Staphylococcus aureus and Streptococcus pneumonia. Antioxidant evaluation of methanolic extract and purified fractions against DPPH showed strong % inhibition of the synthetic free radical. The methanolic extract exhibited 87.41 ± 0.54% inhibition whereas fractions showed: F1, 85.45 ± 0.85; F2, 65.78 ± 0.68; F3, 58.61 ± 0.58; F4, 80.76 ± 0.59; F5, 571.29 ± 0.49; F6, 85.28 ± 0.94; and F7, 48.45 ± 0.62% inhibition. Ascorbic acid (standard) was used as a control with 94.88 ± 0.56% inhibition at a maximum concentration of 1000 µg/mL. The α-glucosidase inhibition assay of methanolic extract and purified fractions at a maximum concentration of 1000 µg/mL showed activities as: methanolic extract, 78.21 ± 0.67; F1, 55.01 ± 0.29; F2, 56.10 ± 0.24; F3, 62.44 ± 1.03; F4, 70.52 ± 0.15; F5, 62.18 ± 0.92; F6, 72.68 ± 0.2; and F7, 57.33 ± 0.05% inhibition. α-Amylase % inhibition of methanolic extract and purified fractions were noted as: methanolic extract, 77.98 ± 0.57; F1, 79.72 ± 0.02; F2, 79.72 ± 0.02; F3, 82.16 ± 0.48; F4, 77.37 ± 0.28; F5, 72.14 ± 0.30; F6, 74.24 ± 0.29; and F7, 56.58 ± 0.10 at the highest concentration of 1000 µg/mL. Acarbose (standard) showed 87.65 ± 0.71% inhibition of α-glucosidase and 85.99 ± 0.44% inhibition of α-amylase at the highest concentration of 1000 µg/mL. It was found that all biological activities of methanolic extract and purified fractions might be attributed to the fact that they are rich sources of phenolic and flavonoids along with other bioactive compounds. The total phenolic and flavonoid contents of methanolic extract were recorded higher as compared to purified fractions (TPC = 70% and TFC = 69%). Amongst the purified fractions, fraction 6 exhibited the highest TPC value (64%), and purified fraction 1 exhibited the highest value of TFC (58%). Recent research demonstrated that Cydonia oblonga may be considered an antibacterial medicinal plant. The result of the present study revealed that it might be utilized for the isolation of bioactive phytochemicals that can lead to new opportunities in the discovery of new antibiotics.

A colorimetric sensing strategy based on enzyme@metal-organic framework and oxidase-like IrO2/MnO2 nanocomposite for α-glucosidase inhibitor screening.

A highly sensitive colorimetric sensing strategy based on enzyme@metal-organic framework (GAA@Cu-MOF) and IrO2/MnO2 nanocomposite was exploited innovatively for screening of α-glucosidase (GAA) inhibitors. IrO2/MnO2 nanocomposite exhibits excellent oxidase-mimicking activity which can directly catalyze the oxidation of 3,3,5,5,-tetramethylbenzidine (TMB) into a blue product with an absorption maximum at 652 nm. And GAA@Cu-MOF can decompose L-ascorbic acid-2-O-α-D-glucopyranosyl (AAG) to ascorbic acid (AA). The produced AA can destroy the IrO2/MnO2 nanocomposite and reduce its oxidase-like activity. However, the generation of AA is restricted when GAA inhibitors are added to the system, which allows the oxidase-like activity of the IrO2/MnO2 nanocomposite to be maintained. In view of this, a method for screening of GAA inhibitors was developed. In addition to enhancing the stability of GAA, the method can also effectively avoid the potential interference of H2O2 in the screening process of GAA inhibitors, which helps to improve the sensitivity of the method. Therefore, highly sensitive determination for acarbose and ascorbic acid are achieved with detection limits of 6.27 nM and 1.23 µM, respectively. The proposed method was successfully applied to screen potential GAA inhibitors from oleanolic acid derivatives. Graphical abstract.

Hydrophilic Interaction Chromatography Coupled with Charged Aerosol Detection for Simultaneous Quantitation of Carbohydrates, Polyols and Ions in Food and Beverages.

Here, we report an accurate and versatile method for the simultaneous determination of 17 sugars (arabinose, erythrose, fructose, galactose, glucose, isomaltulose, lactose, lyxose, maltose, maltotriose, mannose, raffinose, rhamnose, ribose, sucrose, sorbose and xylose), seven polyols (erythritol, inositol, lactitol, maltitol, mannitol, sorbitol and xylitol), five ions (K+, Br-, Cl-, NO3- and SO42-) and the pseudosaccharide acarbose. For compound separation, hydrophilic interaction chromatography (HILIC) coupled to a corona charged aerosol detector (CAD) was used. The method was validated for linearity, precision, reproducibility, retention factor and optimal injection volume. Standards were measured in the range of 1-1000 mg L-1 and showed good intraday and interday repeatability, as well as precision (relative standard deviation (RSD) < 5%). The LODs and LOQs for the 30 analytes were in the range of 0.032-2.675 mg L-1 and 0.107-8.918 mg L-1, respectively. This method exhibited correlation coefficients of at least R2 > 0.97 for all analytes. The method was tested in 24 food and beverage samples to validate the separation efficiency and sensitivity in natural food matrices and to show the practicability of its use for routine food analysis.

Determination of acarbose by capillary zone electrophoresis.

Acarbose (Glucobay, Bayer AG) acts as a potent alpha-glucosidase-inhibitor, which delays the intestinal starch digestion resulting in a reduction of postprandial blood glucose and insulin levels. Acarbose is a pseudo-tetrasaccharide, with two D-glucose units linked via an alpha 1-->4 glycosidic bond to acarviosin, which is a N-glycoside composed of an unsaturated cyclitol and 4-amino-4,6-dideoxy-alpha-D-glucopyranose. Several methods for the determination of acarbose by capillary electrophoresis can be found in literature. They are based either on the derivatisation with 7-aminonaphthalene-1,3-disulfonic acid (ANDS) or on the detection of the unsaturated cyclitol at wavelengths below 200 nm. The aim of our work was the determination of acarbose making use of a previously developed method based on reductive amination with S-phenylethylamine. The aminoalditols generated in the reaction formed differently charged borate-complexes depending on the configuration of the sugar. After successful method optimisation we were able to separate two potential impurities of acarbose, D-maltose und D-glucose. For the quantitation of acarbose in Glucobay tablets an additional borate-buffer system was established, reducing the total time of analysis to less than 10 min.

Utility of an oxidation reaction for the spectrophotometric determination of acarbose in controlled release tablets at various simulated gastrointestinal media.

A sensitive kinetic method for spectrophotometric determination of acarbose is developed and validated for the determination of the drug in bulk and pharmaceutical formulations. The drug was estimated in simulated gastrointestinal media i.e., 0.1 M HCl (pH 1.2) and phosphate buffer (pH 6.8). The method involves the oxidation of acarbose by treating it with a strong oxidizing agent (potassium permanganate (1 x 10(-2) M)) in alkaline media. The reaction kinetics was determined for 20 min at room temperature. The reaction followed first order kinetics and the absorbance of the corresponding manganate ions produced was determined at 610 nm. The absorbance-concentration plot was found to be rectilinear over the concentration range of 2-20 microg/mL. The proposed method was used for estimation of the drug in a novel controlled release dosage form. Thus, the method developed was simple, reproducible and can be successfully applied for the determination of the drug in simulated gastrointestinal fluid.

Inhibitory activity and mechanism of guavinoside B from guava fruits against α-glucosidase: Insights by spectroscopy and molecular docking analyses.

Guavinoside B (GUB) is the main active substance in guava fruit and shows promising biological activities. In this study, the inhibitory activity and mechanism of GUB on α-glucosidase were studied by using spectroscopic techniques, kinetic analysis, and molecular docking. Results indicated that GUB possessed significant inhibition ability on α-glucosidase, which was about 10 times that of acarbose. The GUB was a mixed-type inhibitor, which suppressed the activity of α-glucosidase through a reversible process. Fluorescence analysis revealed that GUB quenched the fluorescence of α-glucosidase statically, the formation of GUB-α-glucosidase complex was a spontaneous and exothermic process, van der Waals forces, hydrogen bonding, and hydrophobic interaction were the predominant driving forces, only one single-binding site on α-glucosidase was involved in the binding process. GUB inserted into the hydrophobic pocket of α-glucosidase with 11 hydrogen bonds and two π-π stacking formed. The presence of GUB changed the microenvironment near the fluorescent amino acids of α-glucosidase, and the structure of α-glucosidase was slightly changed, eventually leading to the decrease of α-glucosidase activity. PRACTICAL APPLICATIONS: Diabetes mellitus (DM) is a worldwide chronic metabolic disease threatening human health seriously. Guava fruit is a popular fruit, and its extracts were reported to show many biological activities. GUB is the main benzophenone glycoside in guava fruits. However, the inhibitory activity and mechanism of its specific active compound GUB are still unclear. Studies have shown that GUB could reversibly inhibit the activity of α-glucosidase, and its inhibitory ability was about 10 times that of acarbose. The kinetics and mechanism of inhibition were revealed. These will facilitate the further research and application of guava fruit and GUB in functional and healthy foods against hyperglycinaemia or even DM.

Simultaneous determination of substrate and product in the process of preparation of valienamine by capillary zone electrophoresis.

A simple and rapid CZE method was established for the simultaneous determination of valienamine, acarbose and validamycin A, using a 20-kV CZE with the detection wavelength of 193 nm and 50 mM phosphoric acid-20 mM Tris (pH 5.3) as a running buffer. The calibration curves of valienamine, acarbose, and validamycin A showed a good linear relationship at a concentration range of 5-1000 microg/mL. The detection limits of valienamine, acarbose, and validamycin A were 0.3, 0.6, and 0.6 microg/mL, respectively, and the average recoveries of each of the above were 99.9, 99.5, and 100.3%. The method has been successfully applied for simultaneous determination of substrate and product in the process of preparation of valienamine.

Development and validation of HPLC-UV-MS method for the control of four anti-diabetic drugs in suspected counterfeit products.

An HPLC-UV method has been developed for the determination of valibose, miglitol, voglibose and acarbose, the four anti-diabetic drugs. The separation was accomplished successfully by using reversed phase chromatography (Prevail carbohydrate column, 250 mm x 4.6 mm, 5 microm) with a gradient acetonitrile-phosphate buffer solution (pH 8.0) at a wavelength of 210 nm. Furthermore, the method of a high-performance liquid chromatography coupled with ESI-MS in positive ionization mode has been established. These two methods were successfully applied to the assay and qualitative detection of four alpha-glucosidase inhibitors in the potential counterfeit anti-diabetic drugs.

Acarbose improves health and lifespan in aging HET3 mice.

To follow-up on our previous report that acarbose (ACA), a drug that blocks postprandial glucose spikes, increases mouse lifespan, we studied ACA at three doses: 400, 1,000 (the original dose), and 2,500 ppm, using genetically heterogeneous mice at three sites. Each dose led to a significant change (by log-rank test) in both sexes, with larger effects in males, consistent with the original report. There were no significant differences among the three doses. The two higher doses produced 16% or 17% increases in median longevity of males, but only 4% or 5% increases in females. Age at the 90th percentile was increased significantly (8%-11%) in males at each dose, but was significantly increased (3%) in females only at 1,000 ppm. The sex effect on longevity is not explained simply by weight or fat mass, which were reduced by ACA more in females than in males. ACA at 1,000 ppm reduced lung tumors in males, diminished liver degeneration in both sexes and glomerulosclerosis in females, reduced blood glucose responses to refeeding in males, and improved rotarod performance in aging females, but not males. Three other interventions were also tested: ursolic acid, 2-(2-hydroxyphenyl) benzothiazole (HBX), and INT-767; none of these affected lifespan at the doses tested. The acarbose results confirm and extend our original report, prompt further attention to the effects of transient periods of high blood glucose on aging and the diseases of aging, including cancer, and should motivate studies of acarbose and other glucose-control drugs in humans.

Ratiometric fluorescence monitoring of α-glucosidase activity based on oxidase-like property of MnO2 nanosheet and its application for inhibitor screening.

In recent years, α-glucosidase (α-Glu) inhibitor has been widely used in clinic for diabetic and HIV therapy. Although different systems have been constructed for sensitive and selective detection of α-Glu and screening its inhibitor, the method based on ratiometric fluorescence for α-glucosidase inhibitor screening remains poorly investigated. Herein, we constructed a new MnO2 nanosheet (NS)-based ratiometric fluorescent sensor for α-glucosidase activity assay and its inhibitor screening. MnO2 NS as an oxidase-mimicking nanomaterial directly oxidized o-phenylenediamine (OPD) into 2,3-diaminophenazine (DAP) which had a strong fluorescence emission at 575 nm, whereas the fluorescence of Ag nanoclusters (NCs) at 450 nm was then quenched by the generated DAP through inner filter effect (IFE). When 2-O-α-d-glucopyranosyl-l-ascorbic acid (AAG) as α-Glu substrate and α-Glu were introduced into the above system, MnO2 NS would be reduced to Mn2+ and lose the oxidase-like property since ascorbic acids (AA) were released with the hydrolysis of AAG by α-Glu. Thus, DAP would not be produced and IFE was stopped accompanying with the fluorescence decrease of DAP and fluorescence increase of AgNCs. A ratiometric fluorescent α-Glu nanosensor was thus developed. The fluorescence intensity ratio of DAP to AgNCs linearly decreased with the increasing of α-Glu concentrations in the range of 0.2-8 U mL-1, and limit of detection was 0.03 U mL-1. This proposed sensing approach was also expanded to α-Glu inhibitor screening and showed excellent applicability. As a typical α-Glu inhibitor, acarbose was investigated with a low detection limit of 10-8 M. The constructed sensor platform was proven to be sensitive and selective as well as simple, label-free and low-cost, making it promising for the accurate diagnosis of relevant disease and discovery of potential drugs.

Optimization of Extraction of Hypoglycemic Ingredients from Grape Seeds and Evaluation of α-Glucosidase and α-Amylase Inhibitory Effects In Vitro.

Grape seeds full with oil and also rich in hypoglycemic ingredients. This study is to improve the comprehensive utilization of grape seeds and the quality of grape seeds oil. Extract water soluble active substances before extracting oil from grape seeds, which show the grape seeds in water soluble extracts containing TP, TC, PC contents, respectively, are 171.1 ± 1.1, 658.8 ± 0.2, 482.3 ± 0.2 (mg GSAE/g). Also we found GSAE samples containing catechin and epicatechin were 44.12 ± 0.21 mg/mL, 111.23 ± 1.29 mg/g, GSAE against α-glucosidase IC50 was 25.25 ± 0.53 g/mL and GSAE against α-amylase IC50 was 66.68 ± 1.1 g/mL, both were competitive way, the effects of Inhibitory were obviously better than that of acarbose. In addition, our process guarantee the fat ingredients remained in the grape seeds and not reduce functional components of oil, our study suggests that GSAE can be used to develop functional foods for prevention and treatment of diabetes and its complications. PRACTICAL APPLICATION: Grape seeds, by-products of brewing wines, are rich in polyphenol and other ingredients. The optimized extraction, composition of constituents and hypoglycemic activity were investigated in this study. The results showed that grape seeds were rich in anthocyanins and polyphenols and other active substances, inhibited α-glucosidase and α-amylase activity, which provide background and practical knowledge for the deep-processed products of grape seeds with high added value.

Quantitation of Alpha-Glucosidase Activity Using Fluorinated Carbohydrate Array and MALDI-TOF-MS.

Quantitation of alpha-glucosidase (α-GD) activity is of significance to diagnosis of many diseases including Pompe disease and type II diabetes. We report here a new method to determine α-GD activity using matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) in combination with carbohydrate microarray and affinity surface chemistry. Carbohydrate probes are synthesized for capture of the enzymatic reaction products and the adducts are loaded onto a fluorinated gold surface to generate an array, which is followed by characterization by MALDI-TOF-MS. The ratio of intensities is used to determine the level of activity of several enzymes. In addition, half maximal inhibitory concentration (IC50) of acarbose and epigallocatechin gallate are also determined using this approach, and the results agree well with the reported values. This method is advantageous as compared to conventional colorimetric techniques that typically suffer matrix interference problems from samples. The use of the polyfluorinated surface has effectively suppressed the interference.

Oligosaccharide sensing with chromophore-modified curdlan in aqueous media.

A newly synthesized chromophore-modified curdlan functions as a saccharide chemosensor in aqueous solution, enabling us to discriminate tetrasaccharide acarbose from 24 mono-, di-, tri-, and tetrasaccharides.