The objective of this study was to evaluate the alpha-amylase inhibitory activity of the methanol extract of Salvia nemorosa against porcine pancreatic amylase in vitro. The plant, sourced from a city in Bihar, was extracted using methanol through maceration for 48 hours. Different concentrations (5, 10, 25, and 50 μg/ml) of the methanol extract were prepared and subjected to alpha-amylase inhibitory assay using starch azure with 0.5 M Tris-HCl buffer and 0.01 M CaCl₂ as the substrate. The methanol extract showed significant alpha-amylase inhibitory activity, with an IC₅₀ value of 4.4 μg/ml. The inhibition increased with dose, and when compared with acarbose, a standard drug for the treatment of diabetes, Salvia nemorosa exhibited greater inhibitory potential. These findings suggest that Salvia nemorosa could be a promising candidate for diabetes management; however, further in vivo studies and clinical trials are needed to confirm its therapeutic potential.

Comparative evaluation of amylases in the oral phase of the INFOGEST static simulation of oro-gastric digestion.

Kinetics and molecular modeling studies on the inhibition mechanism of GH13 α-glycosidases by small molecule ligands

Functional foods have gained popularity in recent decades. They are exploited for their bioactive compounds like polyphenols, which are highly demanded in cosmetic, pharmaceutical and nutraceutical industries. However, extractive techniques and conditions used up to recently are almost obsolete and must be optimized for higher efficiency. The current study aimed to evaluate the antidiabetic potential of an optimized extract of Abelmoschus esculentus (okra) seeds. The optimal conditions for extracting polyphenolic compounds from okra seeds were determined using Microwave Assisted Extraction (MAE). A Face Center Composite Design (FCCD) was used for optimization. Solvent/dry matter ratio, wavelength and time were considered while the response studied was the polyphenolic content. The extract obtained at optimal conditions was characterized using Thin Layer Chromatography (TLC) and Fourier Transform Infra-Red (FTIR) spectroscopy, then tested for its antioxidant, alpha amylase inhibitory and antidiabetic activities. Response Surface Methodology (RSM) permitted the determination of the optimal conditions for phenols extraction as: microwave power 330 W, with a solvent ratio of 97.04/1 mL/g for 9.5 min of extraction time. The optimized extract showed a phenolic content up to 86.37 ± 1.13 mg GAE/g containing quercetin and catechin as revealed by the TLC. Functional groups characteristic of polyphenols were identified on FTIR spectra, and the extract exhibited good in vitro antioxidant capacities with DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical scavenging capacity and FRAP (Ferric Reducing Antioxidant Power Assay). An IC50 of 3.99 ± 0.15 μg/mL was obtained with the DPPH scavenging test. Alpha amylase inhibitory assay revealed that the optimized okra extract behaved as a non-competitive inhibitor of porcine pancreatic amylase with an IC50 of 484.17 ± 2.33 μg/mL. Antidiabetic activity of the extract was observed in streptozotocin-induced diabetic males Wistar rats, as shown by the fasting blood glucose levels, food intake, changes in body weight and serum lipid profile among others.

Trichosanthes kirilowii Maxim taxonomically belongs to the Cucurbitaceae family and Trichosanthes genus. Its whole fruit, fruit peel, seed and root are widely used in traditional Chinese medicines. A ribosome-inactivating protein with RNA N-glycosidase activity called Trichosanthrip was isolated and purified from the seeds of T. kirilowii in our recent previous research. To further explore the biological functions of Trichosanthrip, the cDNA of T. kirilowii alpha-amylase inhibitor (TkAAI) was cloned through rapid-amplification of cDNA ends and its sequence was analyzed. Also, the heterologous protein was expressed in Escherichia coli and its alpha-amylase activity was further measured under optimized conditions. The full-length cDNA of TkAAI was 613bp. The speculated open reading frame sequence encoded 141 amino acids with a molecular weight of 16.14kDa. Phylogenetic analysis demonstrated that the Alpha-Amylase Inhibitors Seed Storage domain sequence of TkAAI revealed significant evolutionary homology with the 2S albumin derived from the other plants in the Cucurbitaceae group. In addition, TkAAI was assembled into pET28a with eGFP to generate a prokaryotic expression vector and was induced to express in E. coli. The TkAAI-eGFP infusion protein was proven to exhibit alpha-amylase inhibitory activity against porcine pancreatic amylase in a suitable reaction system. Analysis of gene expression patterns proved that the relative expression level of TkAAI in seeds is highest. The results presented here forecasted that the TkAAI might play a crucial role during the development of T. kirilowii seeds and provided fundamental insights into the possibility of T. kirilowii derived medicine to treat diabetes related diseases.

Copy number variation (CNV) of the AMY gene in humans has been enthusiastically studied for its association with starch digestibility and obesity. The alpha-amylase (AMY) is a major starch digestive enzyme in mammals. This study aimed to determine the association between CNV of the porcine pancreatic amylase (AMY2B) gene and feed efficiency. Improvement of feed efficiency in growing pigs is of great economic interest. We assayed the copy number of AMY2B by using real-time quantitative PCR (qPCR) in a Large White pig population. We identified three genotypes for AMY2B CNVs, namely I/I (homozygotes of haplotype I; a chromosome with one copy of AMY2B), I/II (heterozygotes of haplotype I and II; a chromosome with two copies) and II/II (homozygotes of haplotype II). We tested the genotypes of the parental generation consisting of six males, 21 females and 265 offspring piglets to validate the AMY2B CNV genotyping. With very few mistyping exceptions, copy numbers of AMY2B were transmitted to piglets in segregation ratios following Mendelian inheritance. Finally, we performed an association analysis between the CNV of the AMY2B gene and feed efficiency traits in 207 uncastrated male pigs. The generalised linear model analysis showed the significant effects of AMY2B CNV genotype on average daily feed intake, total feed intake and feed conversion ratio during growth from 30 kg to 100 kg body weight. However, it was not associated with average daily gain, backfat thickness and loin eye muscle area. Individuals with the genotype I/I had about 76.6 ± 27.1 g lower average daily feed intake, 5.35 ± 1.90 kg lower total feed intake and 0.089 8 ± 0.026 5 lower feed conversion ratio than individuals with I/II and II/II genotypes. Thus, AMY2B CNV has the potential to be an effective genetic marker that could reduce feed costs for pig farming.

Background: Sulfamerazine, a sulfonamide, has been routinely used to treat various bacterial infections, namely Pneumonia, Urinary tract infections, Shigellosis, Bronchitis, Prostatitis, and many more. It interferes with the bacterial folic acid biosynthesis, albeit higher eukaryotes are not susceptible to its action due to the inherent absence of this specific pathway. Objective: In spite of its constant use, Sulfamerazine administration evokes serious issues like the development of antibacterial resistance through environmental contamination, although how it affects the eukaryotic system, specifically its target identification, has not been addressed in detail. Methods: The source of the cell line, including when and from where it was obtained. Whether the cell line has recently been authenticated and by what method. Whether the cell line has recently been tested for mycoplasma contamination. Hela Cells are cultured as per the standard method, amylase and lactate dehydrogenase assay are conducted using a standard procedure with a spectrophotometer. Binding thermodynamics and conformational study have been estimated with isothermal titration calorimetry as well as with docking. Results: Experimental observations reveal that Sulfamerazine inhibits porcine pancreatic amylase in a noncompetitive mode (IC50 of 0.96 mM). The binding of the drug to porcine pancreatic amylase is entropy-driven with conformational changes of the protein as indicated by concomitant redshift. It enhances the inhibitory effects of acarbose and cetapin on their in vitro pancreatic amylase activity. It augments lipid peroxidation and promotes lactic acidosis in a dose-dependent manner. Docking studies ensure effective interactions between Sulfamerazine and proteins like lactic dehydrogenase and porcine pancreatic amylase. Conclusion: Detailed study is to be conducted to confirm whether the molecular scaffold of Sulfamerazine might serve as an effective repurposed drug acting as a lead molecule to design antidiabetic drugs of future use. Alternatively, it should be prescribed with caution under specific medical situations like diabetes, cancer and hepatic disorders manifesting lactic acidosis to avoid the crisis.

Inhibition of in vitro enzymatic starch digestion by coffee extract

Post-meal hyperglycaemia is considered a prominent therapeutic target to attenuate the progression of diabetes and its associated complications. The present study identified fruit extract of Diospyros lotus Linnaeus, of the Ebenaceae family, as an inhibitor of starch digestion through the inhibition of both alpha amylase and alpha glucosidase. The extract inhibits porcine and human pancreatic amylase with IC50 values of 82.5±2.0 and 130.4±24 μg/ml respectively. The inhibition of intestinal sucrase and maltase activity was however considerably weaker. In vitro hydrolysis of solubilised potato starch into glucose yielded comparable inhibition kinetics for 100 μg/ml D. lotus L. extract and 3.5 μM acarbose. Screening the major phenolic constituents revealed that quercetin and myricetin were the strongest alpha amylase inhibitors. D. lotus L. extract showed strong antioxidant activity; however, this provided no meaningful protection against 2-deoxy-ribose induced oxidative stress in INS-1 cells. Taken together these findings identify D. lotus L. fruit as a multi-component functional food with potential to dampen the onset and development of diabetes through the inhibition of post meal hyperglycaemia.

Glycolytic enzyme inhibitory and antiglycation potential of rutin

Retrograded starch is known to be resistant to digestion. We used enzyme kinetic experiments to examine how retrogradation of starch affects amylolysis catalysed by porcine pancreatic amylase. Parallel studies employing differential scanning calorimetry, infra red spectroscopy, X-ray diffraction and NMR spectroscopy were performed to monitor changes in supramolecular structure of gelatinised starch as it becomes retrograded. The total digestible starch and the catalytic efficiency of amylase were both decreased with increasing evidence of retrogradation. A purified sample of retrograded high amylose starch inhibited amylase directly. These new findings demonstrate that amylase binds to retrograded starch. Therefore consumption of retrograded starch may not only be beneficial to health through depletion of total digestible starch, and therefore the metabolisable energy, but may also slow the rate of intestinal digestion through direct inhibition of α-amylase. Such physiological effects have important implications for the prevention and management of type 2 diabetes and cardiovascular disease.

Combating Type-2 diabetes mellitus is a pivotal challenge in front of the present world. Several lines of therapy are in practice for resisting this deadly disease which often culminates with cardiovascular complexities, neuropathy and retinopathy. Among various therapies, administration of alpha glucosidase inhibitors is common and widely practiced. Sulfonylurea category of anti diabetic drug often suffers from cross reactivity with sulfamethoxazole (SMX), a common drug in use to treat a handful of microbial infections. However the specific cellular target generating postprandial hypoglycemia on SMX administration is till date unraveled. The present work has been initiated to elucidate the effects of a group of sulfonamide drugs inclusive of SMX for their amylase inhibitory role. SMX inhibits porcine pancreatic amylase (PPA) in a noncompetitive mode with an average IC50 value 0.94mM respectively. Interaction of SMX with PPA is manifested with gradual quenching of tryptophan fluorescence with concomitant shift in lambda max value (λmax). Binding is governed by entropy driven factor (24.8calmol(-1)K(-1)) with unfavorable contribution from enthalpy change. SMX interferes with the activity of acarbose in a synergistic mode to reduce the effective dose of acarbose as evident from the in vitro PPA inhibition study. In summary, loss of PPA activity in presence of SMX is indicative of structural changes of PPA which is further augmented in the presence of acarbose as explained in the schematic model and docking study.

The effect of fibre and gelatinised starch type on amylolysis and apparent viscosity during in vitro digestion at a physiological shear rate

Aims: α-Amylase and α-glucosidase have been recognized as therapeutic targets for reduction of postprandial hyperglycaemia in diabetes mellitus. Objective of the study was to assess the α-amylase and α-glucosidase inhibitory potential of nine Sri Lankan antidiabetic plants. Study Design: In vitro enzyme inhibitory assays. Place and Duration of Study: Department of Biochemistry, Faculty of Medicine, University of Peradeniya, Sri Lanka, from October 2013 to December 2014. Methodology: Methanol extracts of nine plant parts were used. Pterocarpus marsupium latex was used without extraction. Enzyme inhibition assays were conducted in the presence and absence of plant extracts using porcine pancreatic α-amylase and α-glucosidase from Saccharomyces cerevisiae. Acarbose was used as the standard inhibitor. Percentage inhibition of the two enzymes and the IC50 values were determined. Results: The IC50 values of Ficus racemosa stem bark and Pterocarpus marsupium latex were significantly lower (p< 0.05) than the IC50 value of Acarbose for porcine pancreatic amylase. Lowest IC50 for amylase was observed with P. marsupium. The IC50 values of Phyllanthus emblica fruit, Phyllanthus debilis whole plant, P. marsupium and F. racemosa were significantly lower (p<0.05) than Acarbose for yeast glucosidase. Musa paradisiaca yam and Tinospora cordifolia leaves showed considerable inhibitory effects on glucosidase activity. Coccinia grandis, Gymnema lactiferum, Gymnema sylvestre and Strychnos potatorum seeds did not show considerable inhibitory effects on α-amylase and α-glucosidase. Conclusion: A significantly high (p< 0.05) in vitro α-amylase and α-glucosidase inhibitory activities were observed with the methanol extracts of F. racemosa, P. emblica, P. debilis and P. marsupium.

Background: Biological route for synthesis of copper nanoparticles (CuNPs) with therapeutic potential is a major challenge. In this study, CuNPs were synthesized by D. bulbifera tuber extract (DBTE) which were further evaluated for antidiabetic and free radical scavenging activity. Methods: CuNPs synthesized by DBTE were characterized by UV-visible spectroscopy, transmission electron microscopy, energy dispersive spectroscopy and dynamic light scattering. CuNPs were checked for α-amylase and α-glucosidase inhibition along with interaction studies employing fluroscence spectroscopy, circular dichroism spectroscopy and computational docking. DPPH, nitric oxide and superoxide radical scavenging activities of CuNPs were also checked. Results: Spherical monodispersed CuNPs were synthesized within 5 h that was indicated by a colour change from pale blue to brown. Majority of the nanoparticles synthesized were found to be between 12 to 16 nm as showed in DLS which grew till a final size of 86 to 126 nm as indicated in TEM. Bioreduced CuNPs showed 38.70 ± 1.45% and 34.72 ± 1.22% inhibition against porcine and murine pancreatic amylase, respectively with an uncompetitive mode that was further confirmed by docking studies. Fluorescence spectroscopy confirmed the interaction of CuNPs to the enzyme via Trp residues while CD spectra indicated the structural and conformational changes on binding of CuNPs to the enzyme. CuNPs exhibited 99.09 ± 0.15% inhibition against α-glucosidase while 90.67 ± 0.33% inhibition against murine intestinal glucosidase, respectively. CuNPs showed 40.81 ± 1.44%, 79.06 ± 1.02% and 48.39 ± 1.46% scavenging activity against DPPH, nitric oxide and superoxide radicals respectively. Conclusion: D.bulbifera tuber extract mediated bioreduction is most rapid route to synthesize novel CuNPs with promising antidiabetic and antioxidant properties. This is the first detailed report which provides intense scientific rationale for the use of CuNPs as nanomedicine for efficient control of T2DM and oxidative stress.

Alpha amylase production using microbial source and solid state fermentation has been conducted for past few years in search of thermostable enzyme. Owing to the prolific use of thermostable alpha amylase in various industries like paper, food, detergent, brewing and starch liquefaction process, the production of alpha amylase is still going on. In the present work, Bacillus subtilis (ATCC 6633) has been utilized for generation of alpha amylase followed by optimization of the fermentation media. Change in fermentation conditions like fermentation hour, temperature, inoculums size, nitrogen and sugar sources have pivotal role to enhance alpha amylase yield. The thermal, pH and detergent stability of partially purified alpha amylase have been tested and compared with purified porcine pancreatic amylase. The result is encouraging with approximate 80% retention of alpha amylase activity comparable to purified porcine pancreatic amylase in presence of drastic condition of temperature (60°C), pH (6-11) and detergents. This makes it apt for use in various industries like detergent, food and paper industries.

Competition between humans and livestock for cereal and legume grains makes it challenging to provide economical feeds to livestock animals. Recent increases in corn and soybean prices have had a significant impact on the cost of feed for pig producers. The utilization of byproducts and alternative ingredients in pig diets has the potential to reduce feed costs. Moreover, unlike ruminants, pigs have limited ability to utilize diets with high fiber content because they lack endogenous enzymes capable of breaking down nonstarch polysaccharides into simple sugars. Here, we investigated the feasibility of a transgenic strategy in which expression of the fungal cellulase transgene was driven by the porcine pancreatic amylase promoter in pigs. A 2,488bp 5'-flanking region of the porcine pancreatic amylase gene was cloned by the genomic walking technique, and its structural features were characterized. Using GFP as a reporter, we found that this region contained promoter activity and had the potential to control heterologous gene expression. Transgenic pigs were generated by pronuclear microinjection. Founders and offspring were identified by PCR and Southern blot analyses. Cellulase mRNA and protein showed tissue-specific expression in the pancreas of F1 generation pigs. Cellulolytic enzyme activity was also identified in the pancreas of transgenic pigs. These results demonstrated the establishment of a tissue-specific promoter of the porcine pancreatic amylase gene. Transgenic pigs expressing exogenous cellulase may represent a way to increase the intake of low-cost, fiber-rich feeds.

To assess modifications induced in starch granules during high temperature drying of corn grain and their effect on the nutritive value of corn-starch, physicochemical and structural characteristics of starch granules from corn grains dried at different temperatures have been determined. Additionally, their in vitro digestibility and fermentation patterns were investigated, using a two steps in vitro model of the pig digestive tract. High drying temperatures induced a partial gelatinization of starch granules and produced a very favorable substrate for porcine pancreatic amylase and led to an altered physical structure which affected the rate and extent of starch granules digestion by gastric and pancreatic enzymes. Starch micrographs showed that granules extracted from corn dried at 130°C were less angular, bigger, and had smoother surface than granules extracted from corn dried at lower temperature. High-temperature drying increased the digestibility of wet-milled starch granules, while the residues of starch from corn dried at lower temperature produced higher volume of gas during their in vitro fermentation, despite their more pronounced crystalline characteristic. The residues from pepsin–pancreatic digestion of overall samples analyzed showed highly degraded and pitted granules or fragmented external shells, starch from corn dried at 130°C being the most degraded. Aforementioned changes of nutritional attributes of starch granules are discussed according to the restructuration occurring within both their amorphous and crystalline phase, as well as to the changes of the granules size and purity.

Use of nutritional components from the milk and eventually from the solid feed relates to the growth and development of gastrointestinal tract (GIT). We studied the effect of pancreatic-like enzymes [porcine pancreatic enzymes (Creon) or microbial-derived amylase, protease, and lipase] on GIT morphology and lipid absorption in suckling piglets. Both enzyme preparations, in low or high dose, were fed via a stomach tube twice a day for 7 d starting at 8 d of age and controls received vehicle, n = 6. The day after treatments ended, lipid absorption was tested after which pigs were euthanized and GIT was examined. Enzyme cocktails, irrespective of their origin, increased (P < 0.001) triglyceride level in blood. Enzyme preparation affected (P < 0.001) small intestinal mucosal thickness, villi length, and crypt depth and (P < 0.01) mitotic division of enterocytes. In addition, the external administration of pancreatic enzymes stimulated pancreatic growth as observed by increased (P < 0.05) mitotic division of pancreatic cells. The study revealed that pancreatic or pancreatic-like enzymes of microbial origin administrated in the early postperinatal period enhance GIT development and may be used to better prepare the GIT of piglets for milk use and weaning.

The water extraction and composition of pu-erh tea, as well as the hypoglycemic effect of the water extract of pu-erh tea (WEPT) in vivo and in vitro, are reported to investigate its hypoglycemic effect on diabetes. High-performance liquid chromatography and colorimetric methods are used to analyze the tea catechins, caffeine, polyphenols, amino acids, and polysaccharides of the WEPT. The effect of the WEPT on glucose uptake by cultured HepG2 cells and the inhibition effect of rat intestinal sucrase, maltase, and porcine pancreatic amylase are determined in vitro. Then, the blood glucose and insulin levels of intragastrically administered WEPT on fasting and oral glucose tolerance test (OGTT) using type 2 diabetic db/db (BKS.Cg-m +/+ Lepr(db)/J) mice are determined in vivo. The results showed that the WEPT dose-dependently and significantly increased glucose uptake by HepG2 cells and inhibited rat intestinal sucrase, maltase, and porcine pancreatic amylase activity. The WEPT intragastrically given for 4 weeks suppressed the increase in blood insulin and glucose levels of db/db mice fasted overnight. In OGTT, the WEPT improved impaired glucose tolerance and ameliorated retarded insulin response at 60 and 120 min in db/db mice. These results suggest that the WEPT has beneficial effects on glucose homeostasis in type 2 diabetes and in amendment of insulin resistance.