Synergistic cytotoxicity induced by α-solanine and α-chaconine

α-Solanine and α-chaconine are well-known potato toxins, but the mechanism of the synergistic cytotoxic effect of these alkaloids has been little clarified. This study confirmed their synergistic cytotoxic effects on C6 rat glioma cells by three different cell viability tests, namely WST-1 (water-soluble tetrazolium) assay sensitive to intracellular NADH concentration, menadione-catalysed chemiluminescent assay depending on both NAD(P)H concentration and NAD(P)H:quinone reductase activity, and LDH (lactate dehydrogenase) assay sensitive to the release of LDH from damaged cells. The maximum cytotoxic effect was observed at a ratio of 1:1 between α-solanine and α-chaconine at micromolar concentrations. The cytotoxic effects of these alkaloids were observed immediately after incubation and were constant after 30 min, suggesting that rapid damage of plasma membrane causes the lethal disorder of metabolism.     

Anti-diabetic activity peptides from albumin against α-glucosidase and α-amylase

The objectives of this study were to identify novel peptides from albumin, and to evaluate and validate the anti-diabetic activity of peptides against α-glucosidase and α-amylase. In the research, albumin hydrolysate was purified and identified, tandem MS was adapted to characterise the amino acid sequences of peptides from the hydrolysate. In addition, anti-diabetic effects of the peptides with α-glucosidase and α-amylase inhibitory activity have been performed. The present work found eight novel peptides from albumin. Results also suggested that peptide KLPGF had α-glucosidase inhibitory activity with an IC(50) of 59.5±5.7μmoll(-1) and α-amylase inhibitory activity with an IC(50) of 120.0±4.0μmoll(-1). In conclusion, the results revealed that the peptide KLPGF was a potential anti-diabetic inhibitor.     

Identification and characterization of novel α-amylase and α-glucosidase inhibitory peptides from camel whey proteins

This study explores the inhibitory properties of camel whey protein hydrolysates (CWPH) toward α-amylase (AAM) and α-glucosidase (AG). A general full factorial design (3 × 3) was applied to study the effect of temperature (30, 37, and 45°C), time (120, 240, and 360 min), and enzyme (pepsin) concentration (E%; 0.5, 1, and 2%). The results showed that maximum degree of hydrolysis was obtained when hydrolysis was carried out at higher temperature (45°C; P < 0.05), compared with lower temperatures of 30 and 37°C. Electrophoretic pattern displays degradation of all protein bands upon hydrolysis by pepsin at various hydrolysis conditions applied. All the 27 CWPH generated showed significant AAM and AG inhibitory potential as indicated by their lower IC₅₀ values (mg/mL) compared with intact whey proteins. In total 196 peptides were identified from selected hydrolysates and 15 potential peptides (PepSite score > 0.8; http://pepsite2.russelllab.org/) were explored via in silico approach. Novel peptides PAGNFLMNGLMHR, PAVACCLPPLPCHM, MLPLMLPFTMGY, and PAGNFLPPVAAAPVM were identified as potential inhibitors for both AAM and AG due to their high number of binding sites and highest binding probability toward the target enzymes. CCGM and MFE, as well as FCCLGPVPP were identified as AG and AAM inhibitory peptides, respectively. This is the first study that reports novel AG and AAM inhibitory peptides from camel whey proteins. The future direction for this research involves synthesis of these potential AG and AAM inhibitory peptides in a pure form and investigate their antidiabetic properties in the in vitro, as well as in vivo models. Thus, CWPH can be considered for potential applications in glycaemic regulation.     

Metabolite profiling and inhibitory properties of leaf extracts of Ficus benjamina towards α-glucosidase and α-amylase

The use of antioxidant-rich medicinal plants having the potential to reduce oxidative stress and postprandial hyperglycemic pressure is one of the most promising option for the management of diabetes. This study presents information on metabolite profiling and in vitro anti-diabetic effects of leaf extracts of Ficus benjamina. The DPPH (2, 2-diphenyl-1-picrylhydrazyl radicals) assay was performed to determine the in vitro antioxidant potential of the plant extracts. The anti-diabetic effects were investigated by evaluating inhibitory properties of F. benjamina leaf extracts towards carbohydrate hydrolyzing enzymes, i.e., α-glucosidase and α-amylase, whereas ¹H NMR and UHPLC-QTOF-MS/MS analytical methods were employed for metabolite profiling of F. benjamina leaf extracts. Among 40, 60, 80, and 100% ethanolic leaf extracts of F. benjamina, 80% ethanolic extract exhibited the highest antioxidant activity based upon its DPPH radical scavenging ability (IC₅₀ value: 63.71 ± 2.66 µg/mL). The 80% ethanolic leaf extract of F. benjamina also proved to be the most efficient α-glucosidase and α-amylase inhibitor with IC₅₀ values of 9.65 ± 1.04 µg/mL and 13.08 ± 1.06 µg/mL, respectively; these values were even better than acarbose with α-glucosidase inhibition activity (IC₅₀ = 116.01 ± 3.83 µg/mL) and α-amylase inhibition activity (IC₅₀ = 152.66 ± 7.32 µg/mL). Moreover, a total of 31 metabolites were identified in F. benjamina leaf extract, which may have the potential to contribute to its antioxidant and inhibitory properties against carbohydrate hydrolyzing enzymes. The findings of this study depict F. benjamina leaf extracts as a promising α-glucosidase and α-amylase inhibitor, and therefore, can be utilized for the development of anti-diabetic functional diets/nutra-pharmaceuticals.     

The α-amylase and α-glucosidase inhibitory effects of Irish seaweed extracts

To date, numerous studies have reported on the antidiabetic properties of various plant extracts through inhibition of carbohydrate-hydrolysing enzymes. The objective of this research was to evaluate extracts of seaweeds for α-amylase and α-glucosidase inhibitory effects. Cold water and ethanol extracts of 15 seaweeds were initially screened and from this, five brown seaweed species were chosen. The cold water and ethanol extracts of Ascophyllum nodosum had the strongest α-amylase inhibitory effect with IC₅₀ values of 53.6 and 44.7 μg/ml, respectively. Moreover, the extracts of Fucus vesiculosus Linnaeus were found to be potent inhibitors of α-glucosidase with IC₅₀ values of 0.32 and 0.49 μg/ml. The observed effects were associated with the phenolic content and antioxidant activity of the extracts, and the concentrations used were below cytotoxic levels. Overall, our findings suggest that brown seaweed extracts may limit the release of simple sugars from the gut and thereby alleviate postprandial hyperglycaemia.     

Identification and characterization of yak α-lactalbumin and β-lactoglobulin

α-Lactalbumin (α-LA) and β-lactoglobulin (β-LG) were isolated from yak milk and identified by mass spectrometry. The variant of α-LA (L8IIC8) in yak milk had 123 amino acids, and the sequence differed from α-LA from bovine milk. The amino acid at site 71 was Asn (N) in domestic yak milk, but Asp (D) in bovine and wild yak milk sequences. Yak β-LG had 2 variants, β-LG A (P02754) and β-LG E (L8J1Z0). Both domestic yak and wild yak milk contained β-LG E, but it was absent in bovine milk. The amino acid at site 158 of β-Lg E was Gly (G) in yak but Glu (E) in bovine. The yak α-LA and β-LG secondary structures were slightly different from those in bovine milk. The denaturation temperatures of yak α-LA and β-LG were 52.1°C and 80.9°C, respectively. This study provides insights relevant to food functionality, food safety control, and the biological properties of yak milk products.     

Enrichment of dry-cured ham with α-linolenic acid and α-tocopherol by the use of linseed oil and α-tocopheryl acetate in pig diets

The use of α-linolenic acid and α-tocopherol enriched pork on the fatty acids and the sensory characteristics of Spanish dry-cured hams have been studied. Five batches of hams were manufactured using the posterior legs of pigs fed on diets with the same ingredients except for the oil source: sunflower (C), linseed (L) or linseed and olive (1/1, w/w, LO). Two different α-tocopheryl acetate concentrations [20 (C, L and LO) or 220 (LOE and LE)mg/kg diet] were used. Biceps femoris and Semitendinosus/Semimembranosus muscles from hams with low polyunsaturated fatty acid n-6/n-3 ratio (less than 3) were obtained from animals fed on linseed and linseed/olive oil enriched diets. However, hams from animals fed on diets added with linseed and α-tocopheryl acetate (20mg/kg diet) (batch L) were rejected by consumers because of less acceptable sensory characteristics and higher TBARs. The remaining hams had satisfactory sensory and nutritional characteristics.     

In Vitro Potential of Ascophyllum nodosum Phenolic Antioxidant-Mediated α-Glucosidase and α-Amylase Inhibition

ABSTRACT: Ascophyllum nodosum is a brown seaweed that grows abundantly in the Northeast coastal region. In this study, the potential of A. nodosum for type 2 diabetes management through antioxidant-mediated α-glucosidase and α-amylase inhibition was investigated. After the initial screening of 4 locally harvested seaweeds, A. nodosum was chosen for its highest phenolic content and was subjected to water extraction. Among extraction ratios of 50 g to 100 to 1000 mL at room temperature, 50 g/400 mL yielded the highest phenolic content of 4.5 mg/g wet weight. For evaluation of extraction temperature ranging from 20 to 80 °C, 50 g/400 mL was chosen as a minimum amount of extractant. Among temperatures studied, extraction at 80 °C resulted in the highest total phenolic contents (4.2 mg/g wet weight). All extracts had similar levels of antioxidant activity in the range of 60% to 70% in terms of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity. The 80 °C extract had the highest α-glucosidase and α-amylase inhibitory activity with IC₅₀ of 0.24 and 1.34 μg phenolics, respectively, compared to the IC₅₀ of acarbose, reference inhibitor, being 0.37 and 0.68 μg. The results show that fresh A. nodosum has strong α-glucosidase and mild α-amylase inhibitory activities that correlated with phenolic contents. This study suggests a nutraceutical potential of A. nodosum based on phytochemical antioxidant and antihyperglycemia activities.     

Effect of dietary α-tocopherol supplementation and gamma-irradiation on α-tocopherol retention and lipid oxidation in cooked minced chicken

The effects of dietary α-tocopherol supplementation and gamma-irradiation on α-tocopherol retention and lipid oxidation in cooked minced chicken during refrigerated storage were studied. Minced breast and thigh meat from broilers fed diets supplemented with 100, 200 or 400 mg α-tocopheryl acetate/kg feed was irradiated at 2.5 or 4.0kGy. Cooked irradiated and unirradiated meat was stored at 4 °C for 5 days. α-Tocopherol concentrations increased with increasing dietary supplementation. Concentrations decreased during storage, but retention was not affected by irradiation. Lipid stability was determined by measuring the formation of thiobarbituric acid-reacting substances (TBARS) and cholesterol oxidation products (COPs) during storage. TBARS and COPs increased during storage and were reduced by increasing levels of dietary α-tocopheryl acetate supplementation. Irradiation accelerated TBARS formation during storage, but this was prevented by supplementation with 200 mg α-tocopheryl acetate/kg feed. Irradiation tended to increase COPs during storage, although no consistent effects were observed. In general supplementation with over 400 mg α-tocopheryl acetate/kg feed may be required to control cholesterol oxidation in minced chicken. The results suggest that, overall, irradiation had little effect on lipid stability in α-tocopherol-supplemented meat following cooking and storage.     

Uptake of α-tocopherol in porcine plasma and tissues

The effect of feeding α-tocopheryl acetate to pigs on the rate and extent of uptake of α-tocopherol in various tissues was investigated. The rate of iron ascorbate-induced lipid peroxidation was also studied. One hundred and eight Landrace × Large White pigs were assigned at random to one of the following barley-based diets: 20 mg α-tocopheryl acetate/kg feed for up to 126 days; 20 mg/kg feed to day 91, followed by 200 mg/kg feed to day 126, or 200 mg/kg feed for up to 126 days: Pigs from each group were slaughtered at specified intervals. For pigs fed the diet supplemented with 200 mg α-tocopheryl acetate/kg feed, α-tocopherol levels increased with increasing supplementation time up to day 126 in all tissues studied. The highest levels of α-tocopherol were observed in kidney fat and subcutaneous fat (inner layer) followed by subcutaneous fat (outer layer), liver, lung, heart, kidney, with muscle and brain containing approximately the same level. The α-tocopherol concentrations in all tissues examined, from pigs fed 200 mg α-tocopheryl acetate/kg feed from weaning to day 126 were greater than those fed the supplemented diet for 35 days. Iron-induced lipid peroxidation was reduced by dietary α-tocopheryl acetate supplementation, and muscle samples from pigs supplemented for 126 days were significantly (p < 0.05) less susceptible to peroxidation compared to muscle from pigs fed the same supplemented diet for 35 days.     

The inhibitory effects of ethanol extracts from sorghum,foxtail millet and proso millet on α-glucosidase and α-amylase activities

Cereal crops have recently experienced increased interest due to their potential health benefits. It has been suggested that the intake of whole grain foods is beneficial to the prevention and management of diabetes mellitus. In this study, we investigated the inhibitory effect of 70% EtOH extracts from different cultivars of sorghum, foxtail millet and proso millet on α-glucosidase and α-amylase. Among the six sorghum cultivars, Mongdang-susu(SS-1), Me-susu(SS-2), Susongsaengi-susu(SS-3) and Sikyung-susu(SS-4) extracts exhibited higher inhibitory activities against α-glucosidase (IC₅₀ = 1.1–1.4 μg/ml) than acarbose, reference inhibitor (IC₅₀ = 2.1 μg/ml). In addition, these extracts strongly inhibited degradation of starch by pancreatic and salivary α-amylase, whereas extracts from foxtail and proso millets exhibited no visible or detectible inhibitory effect on α-amylase or on α-glucosidase activity. These in vitro studies indicate the potential of sorghum in the development of effective anti-diabetic agents.     

Activity and stability enhancement of α-amylase treated with sub- and supercritical carbon dioxide

Various physical, chemical and genetic approaches have been applied in order to enhance enzyme stability and activity. In this study, the aim was to investigate the capability of sub- and supercritical carbon dioxide to alter the stability and activity of α-amylase as an alternative technique. The effects of operational parameters such as pressure (50-300 bar), temperature (28-80 °C), CO? flow (2-10 g min?1) and time (60-180 min) were evaluated in regard to the activity and stability of fungal based α-amylase from Aspergillus oryzea. The activity of untreated enzyme was determined as 17,726 μmol/ml/min. While both sub- and supercritical conditions enhanced the activity, the increase in flow rate had an adverse effect and the activity was decreased by 28.9% at a flow rate of 10 g min?1 under supercritical conditions. Nuclear magnetic resonance (NMR) spectra of untreated enzyme and treated samples exhibiting the lowest and the highest activities were almost identical except for the chemical shifts observed at the lowest activity sample from 4.0 to 4.4 ppm which were assigned to protons of hydrogen-bonded groups. Optimum conditions were determined as 240 bar, 41 °C, 4 g min?1 CO? flow and 150 min of process duration yielding 67.7% (29,728 μmol/ml/min) higher activity than the untreated enzyme providing fundamental basis for enzymatic applications.     

α-Glucosidase and α-amylase inhibitory activities of guava leaves

The 75% ethanol extract from guava (Psidium guajava Linn.) leaves was extracted further, in turn, with CH₂Cl₂, EtOAc and n-BuOH to afford four fractions, CH₂Cl₂-soluble, EtOAc-soluble, n-BuOH-soluble and residual extract fractions. Both the n-BuOH-soluble and EtOAc-soluble fractions showed high inhibitory activity against α-glucosidase and α-amylase. Seven pure flavonoid compounds, quercetin (1), kaempferol (2), guaijaverin (3), avicularin (4), myricetin (5), hyperin (6) and apigenin (7), were isolated (using enzyme assay-guide fractionation method) from the n-BuOH-soluble and EtOAc-soluble fractions. The structures of these pure compounds were determined on the basis of MS and NMR data and the activities of these compounds were evaluated. Compounds 1, 2 and 5 showed high inhibitory activities, with IC₅₀ values of 3.5 mM, 5.2 mM and 3.0 mM against sucrase, with IC₅₀ values of 4.8 mM, 5.6 mM and 4.1 mM against maltase and with IC₅₀ values of 4.8 mM, 5.3 mM and 4.3 mM against α-amylase, respectively. We found that myricetin showed the most powerful activity among these compounds with a 70% inhibition against sucrase at a concentration of 1.5 mg/ml. The hydroxyl group at the 3-position on the A-ring and a number of hydroxyl groups attached to the C-ring played important roles in the inhibition activity. There was an obvious synergistic effect (the mixing action of two compounds) against α-glucosidase, but against α-amylase this was not found. This is the first study of the active compositions of guava leaves and the biological activity of the active compositions against α-glucosidase and α-amylase.     

Oxidative stability enhancement of broiler bird meats with α-lipoic acid and α-tocopherol acetate supplemented feed

Depression of meat quality is known to be caused by lipid peroxidation occurring in meat. Supplementation of antioxidants in feed decreases lipid peroxidation and improves the oxidative stability of meat after slaughtering. The present study demonstrated that meat obtained from broiler birds fed feed supplemented with α-tocopherol acetate (200 mg/kg feed) along with α-lipoic acid (25, 75, or 150 mg/kg feed) exhibited increased oxidative stability and reduced fat content. The total phenolic content and α-lipoic acid content increased in the meat as the concentration of α-lipoic acid supplementation increased. The protein content in the meat was not changed by the supplementation of α-lipoic acid and α-tocopherol acetate. The results of DPPH and TBA assays demonstrated that feed supplemented with α-lipoic acid and α-tocopherol acetate also enhanced the antioxidant activity of broiler meat. On the other hand, the meat from broiler birds fed feed supplemented with oxidised oil (4% in feed) reduced its oxidative stability.     

Purification and characterization of hydrolytic and transgalactosyl α-galactosidase from Lactobacillus helveticus ATCC 10797

α-Galactosidase purified from Lactobacillus helveticus ATCC 10797 by fast performance liquid chromatography system using ion exchange and gel-filtration columns showed the K _m of 3.83 mM and V _max of 416.44 µmol/min/mg protein calculated from the substrate p-nitrophenyl-α-d-galactopyranoside. The molecular mass was 188 kDa by gel-filtration, but 90 kDa by SDS-PAGE, indicating a homodimer. The optimum temperature was 37 °C, and the optimum pH was at 6 with an acceptable stability between pH 4 and 8. This enzyme was activated by 10 mM monovalent ions such as K⁺, NH₄ ⁺, Li⁺, and CS⁺, while the activity was inhibited by divalent ions such as Cu²⁺, Zn²⁺, and Fe²⁺. Melibiose was hydrolyzed to glucose and galactose, raffinose to galactose and sucrose, while stachyose to galactose and sucrose. A novel source of α-galactosidase from L. helveticus possessing both hydrolytic activity to eliminate flatulence sugars and transgalactosylation activities to synthesize galacto-oligosaccharides is identified and characterized.     

Genome-wide association study for αS1- and αS2-casein phosphorylation in Dutch Holstein Friesian

Phosphorylation of caseins (CN) is a crucial post-translational modification that allows caseins to form colloid particles known as casein micelles. Both α_S1- and α_S2-CN show varying degrees of phosphorylation (isoforms) in cow milk and were suggested to be more relevant for stabilizing internal micellar structure than β- and κ-CN. However, little is known about the genetic background of individual α_S2-CN phosphorylation isoforms and the phosphorylation degrees of α_S1- and α_S2-CN (α_S1-CN PD and α_S2-CN PD), defined as the proportion of isoforms with higher degrees of phosphorylation in total α_S1- and α_S2-CN, respectively. We aimed to identify genomic regions associated with these traits using 50K single nucleotide polymorphisms for 1,857 Dutch Holstein Friesian cows. A total of 10 quantitative trait loci (QTL) regions were identified for all studied traits on 10 Bos taurus autosomes (BTA1, 2, 6, 9, 11, 14, 15, 18, 24, and 28). Regions associated with multiple traits were found on BTA1, 6, 11, and 14. We showed 2 QTL regions on BTA1, one affecting α_S2-CN production and the other harboring the SLC37A1 gene, which encodes a phosphorus antiporter and affects α_S1- and α_S2-CN PD. The QTL on BTA6 harbors the casein gene cluster and affects individual α_S2-CN phosphorylation isoforms. The QTL on BTA11 harbors the PAEP gene that encodes for β-lactoglobulin and affects relative concentrations of α_S2-CN-10P and α_S2-CN-11P as well as α_S1-CN PD and α_S2-CN PD. The QTL on BTA14 harbors the DGAT1 gene and affects relative concentrations of α_S2-CN-10P and α_S2-CN-11P as well as α_S1-CN PD and α_S2-CN PD. Our results suggest that effects of identified genomic regions on phosphorylation of α_S1- and α_S2-CN are related to changes in milk synthesis and phosphorus secretion in milk. The actual roles of SLC37A1, PAEP, and DGAT1 in α_S1- and α_S2-CN phosphorylation in Dutch Holstein Friesian require further investigation.     

Immunoreactive properties of α-casein and κ-casein: Ex vivo and in vivo studies

The aim of this study was to evaluate the ex vivo and in vivo studies immune potential of α- and κ-casein. Ex vivo, naïve mouse splenocytes were stimulated with α- or κ-casein. After 120 h of culture, the proliferation index (PI), determined by 3-(4,5 dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and carboxyfluorescein diacetate N-succinimidyl ester (CFSE) staining, did not vary for either antigen, suggesting similar ex vivo immunogenic potential of both casein fractions. In vivo, BALB/ccmdb mice were sensitized with α- or κ-casein and then gavaged with primary antigen. Mice immunized with α-casein had higher levels of IgG (2^16.33) and IgA (2^10.22) in serum at the end of the experiment compared with mice immunized with κ-casein (2¹⁵ and 2^9.3 for IgG and IgA, respectively). The use of α-casein for mouse immunization and ex vivo lymphocyte stimulation resulted in higher concentrations of secreted cytokines (IL-4, IL-10) compared with κ-casein stimulation. This is consistent with increasing regulatory T cell (Treg) lymphocyte populations, independent of the antigen used for stimulation. In summary, the immunogenic potential of α- and κ-casein was similar. Humoral and cellular immune responses confirmed their strong, independent potential to induce B and T cells. We propose that the lymphocyte proliferation index be used as an initial screening for protein immunogenicity.