Exploring the α-amylase-inhibitory properties of tannin-rich extracts of Cytinus hypocistis on starch digestion.

Cytinus hypocistis(L.) L. is an edible parasitic plant that grows within the roots of its host. In addition to its use as famine food in the past, it is also tradidionally used for treating several illnesses such as intestinal problems, inflammations, tumors, and bleeding. This species is rich in hydrolysable tannins, compounds often associated with inhibiting starch digestion. Therefore, the present work investigated how effectively C. hypocistis tannin-rich extracts inhibited enzymes involved in starch digestion and if such effect also occurs in vivo. The latter premise was approached using the starch tolerance test in mice. Two optimized hydroethanolic extracts were used, a heat-assisted and an ultrasound-assisted extract, with known hydrolysable tannin content. Both extracts demonstrated potent inhibition of α-amylase. Inhibitions were of the mixed type with inhibitor constants in the 15 µg/mL range. The inhibition of the intestinal α-glucosidase was at least ten times less effective. The inhibition of the α-amylase was negatively affected by in vitro gastrointestinal digestion and bovine serum albumin. In vivo, both extracts inhibited starch digestion at doses between 100 and 400 mg/mL in healthy mice. The highest doses of the ultrasound and heat extracts diminished the peak glucose levels in the starch tolerance test by 46 and 59.3%, respectively. In streptozotocin diabetic mice, this inhibition occurred only at the dose of 400 mg/mL. Under this condition, diminution of the peak glucose concentration in the starch tolerance test was equal to 36.7% and 48.8% for the ultrasound and heat extracts, respectively. Maltose digestion was not inhibited by the C. hypocistis extracts. Qualitatively and quantitatively, thus, the actions of both extracts were similar. The results allow adding a new biological property to C. hypocistis, namely, the ability to decrease the hyper-glycemic excursion after a starch-rich meal, propitiating at the same time a diminished caloric intake.

The growth performance, antioxidant and immune responses, and disease resistance of Litopenaeus vannamei fed on diets supplemented with Indian ginseng (Withania somnifera).

In the current study, white-leg shrimp (Litopenaeus vannamei) were fed on diets containing varying doses of Withania somnifera aqueous extract (WSAE) at a rate of 0 (control), 0.5, 1.0, and 2.0 g/kg feed for 56 days. After the feeding trial, shrimps in all groups were challenged with the exposure to Vibrio harveyi for ten days during which animals' mortality was observed. It is noted that the dietary WSAE linearly and quadratically stimulated shrimp's growth indices particularly at the treatment of 2.0 g/kg feed. Compared to the control group, the WSAE-fed L. vannamei had significantly higher villi length, villi width, and absorption area particularly in the treatment of 2.0 g/kg feed. Furthermore, L. vannamei fed on WSAE-enriched diets consumed more feed and exhibited higher total proteolytic activity, lipase, and α-amylase activities as compared with the control group. The dietary WSAE at escalating levels linearly and quadratically enhanced the antioxidant activity (serum superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), total antioxidant capacity, and reduced glutathione) and the immune response (total hemocyte counts, total protein, lysozyme, and phagocytic activity). Similarly, the mRNA expression levels of cMn-SOD, CAT, and GPx genes were linearly and quadratically upregulated in the hepatopancreas of L. vannamei fed on WSAE-enriched diets (especially in the 2.0 g/kg feed treatment), while their lowest levels were significantly observed in the control group. On the other hand, malondialdehyde levels were significantly decreased in WSAE-supplemented shrimp groups, and its highest levels were observed in animals fed on the control diet. After the bacterial exposure, the survival rates of L. vannamei fed on 1.0 and 2.0 g WSAE/kg feed (61.3% and 66.7%, respectively) were higher than those in the control animals. Taken together, the results obtained herein indicate that inclusion of WSAE in diets of L. vannamei effectively enhanced the growth, antioxidant biomarkers, immune response, and resistance to the V. harveyi infection, particularly at the treatment of 2.0 g/kg feed.

[Anticancer effect of modified banana (Musa cavendish AAA) starch in rats with 1.2-dimethylhydrazine]. / Efecto anticancerígeno del almidón modificado de banano (Musa cavendish AAA) en ratas con 1,2-dimetilhidrazina.

INTRODUCTION: Introduction: resistant starch (RS) is not completely digested in the human intestine but is fermented in the colon; intestinal pH decreases as short-chain fatty acids are produced. This is beneficial for health, and for preventing and treating rectal colon cancer. Pyrodextrinization and enzymatic hydrolysis are modifications to native starch (NS) that may increase the amount of RS. Objective: the objective of this project was to evaluate the effects of M. cavendish AAA native and both chemically and enzymatically modified starches on tumor markers in rats. Methods: modifications (chemical and enzymatic) were made to M. cavendish AAA NS, and were evaluated in rats with 1,2-DMH. Male Sprague Dawley rats (25) were used, divided into five experimental groups: PC, NC, NS, PI, and ERM. During 4 weeks they received the experimental diet assigned to each group. The PC, NS, PI and ERM groups received 2 weekly s.c. (subcutaneous) injections of 1,2-DMH (40 mg/kg) (third and fourth week). In feces, pH, ß-glucuronidase enzyme, and short-chain fatty acids were evaluated, and a histopathological study was performed of the intestine to detect microscopic lesions. Results: the activity of ß-glucuronidase decreased (p < 0.05) for NS, PI and ERM vs. PC. The highest proportion of butyric acid was observed in the NS (p < 0.05) vs. NC group. Sixty percent of enteritides were severe in grade in the PC group, and 40 % in the experimental groups. Conclusions: native starch granules resisted pyrodextrinization, but treatment with α-amylase broke the structure of the pyrodextrin granule. According to the treatments given to the rats, as the amount of RS present in the diet increases (NS), the neoplastic cells do not advance beyond the basement membrane, suggesting a possible cell-protective or anticancer effect.

INTRODUCCIÓN: Introducción: el almidón resistente (AR) no se digiere completamente en el intestino humano sino que se fermenta en colon; disminuye el pH intestinal, ya que se producen ácidos grasos de cadena corta, interviniendo de manera benéfica en el tratamiento preventivo y curativo del cáncer de colon rectal. La pirodextrinización y la hidrólisis enzimática son modificaciones al almidón nativo (AN) que pueden incrementar la cantidad de AR. Objetivo: el objetivo de este proyecto fue evaluar los efectos del almidón nativo de M. cavendish AAA y de los almidones modificados química y enzimáticamente sobre diversos marcadores tumorales en ratas. Métodos: se realizaron modificaciones (química y enzimática) del AN del banano M. cavendish AAA y se evaluaron en ratas tratadas con 1,2-DMH. Se utilizaron 25 ratas Sprague Dawley machos divididas en cinco grupos experimentales: CP, CN, AN, PI y MER. Durante 4 semanas recibieron la dieta experimental asignada a cada grupo. Los grupos CP, AN, PI y MER recibieron 2 inyecciones s.c. (subcutáneas) semanales de 1,2-DMH (40 mg/kg) (semanas 3 y 4). En las heces se evaluaron el pH, la enzima ß-glucuronidasa y los ácidos grasos de cadena corta, y se realizó un estudio histopatológico del ciego y el colon para detectar lesiones microscópicas. Resultados: la actividad de ß-glucuronidasa disminuyó (p < 0,05) para los grupos AN, PI y MER en comparación con el CP. La mayor proporción de ácido butírico se observó en el AN (p < 0,05) frente al CN. El 60 % de las enteritis fueron de grado severo en el CP, mientras que en los grupos experimentales fueron de 40 %. Conclusiones: los gránulos de almidón nativo resistieron la pirodextrinización pero el tratamiento con α-amilasa rompió la estructura del gránulo de pirodextrina. De acuerdo a los tratamientos suministrados a las ratas, conforme mayor es la cantidad de AR presente en la dieta (AN), las células neoplásicas no avanzan más allá de la membrana basal, sugiriendo un posible efecto protector o anticancerígeno celular.

Marine Peptides as Potential Agents for the Management of Type 2 Diabetes Mellitus-A Prospect.

An increasing prevalence of diabetes is known as a main risk for human health in the last future worldwide. There is limited evidence on the potential management of type 2 diabetes mellitus using bioactive peptides from marine organisms, besides from milk and beans. We summarized here recent advances in our understanding of the regulation of glucose metabolism using bioactive peptides from natural proteins, including regulation of insulin-regulated glucose metabolism, such as protection and reparation of pancreatic ß-cells, enhancing glucose-stimulated insulin secretion and influencing the sensitivity of insulin and the signaling pathways, and inhibition of bioactive peptides to dipeptidyl peptidase IV, α-amylase and α-glucosidase activities. The present paper tried to understand the underlying mechanism involved and the structure characteristics of bioactive peptides responsible for its antidiabetic activities to prospect the utilization of rich marine organism proteins.

Effects of arginine and leucine substitutions on anti-endotoxic activities and mechanisms of action of cationic and amphipathic antimicrobial octadecapeptide from rice α-amylase.

Previously, we showed that the antimicrobial cationic and amphipathic octadecapeptide AmyI-1-18 from rice α-amylase (AmyI-1) inhibited the endotoxic activity of lipopolysaccharide (LPS) from Escherichia coli. In addition, we demonstrated that several AmyI-1-18 analogs containing arginine or leucine substitutions, which were designed on the basis of the helical wheel projection of AmyI-1-18, exhibited higher antimicrobial activity against human pathogenic microorganisms than AmyI-1-18. In the present study, anti-inflammatory (anti-endotoxic) activities of five AmyI-1-18 analogs containing arginine or leucine substitutions were investigated. Two single arginine-substituted and two single leucine-substituted AmyI-1-18 analogs inhibited the production of LPS-induced nitric oxide in mouse macrophages (RAW264) more effectively than AmyI-1-18. These data indicate that enhanced cationic and hydrophobic properties of AmyI-1-18 are associated with improved anti-endotoxic activity. In subsequent chromogenic Limulus amebocyte lysate assays, 50% inhibitory concentrations (IC50 ) of the three AmyI-1-18 analogs (G12R, D15R, and E9L) were 0.11-0.13 µm, indicating higher anti-endotoxic activity than that of AmyI-1-18 (IC50, 0.22 µm), and specific LPS binding activity. In agreement, surface plasmon resonance analyses confirmed direct LPS binding of three AmyI-1-18 analogs. In addition, AmyI-1-18 analogs exhibited little or no cytotoxic activity against RAW264 cells, indicating that enhancements of anti-inflammatory and LPS-neutralizing activities following replacement of arginine or leucine did not result in significant increases in cytotoxicity. This study shows that the arginine-substituted and leucine-substituted AmyI-1-18 analogs with improved anti-endotoxic and antimicrobial activities have clinical potential as dual-function host defense agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Inhibitory effect of chlorogenic acid on digestion of potato starch.

The effect of the chlorogenic acid isomer 5-O-caffeoylquinic acid (5-CQA) on digestion of potato starch by porcine pancreatic alpha amylase (PPAA) was investigated using isolated starch and cooked potato tuber as substrates. In vitro digestion was performed on five varieties of potato with varying phenolic content. Co- and pre-incubation of PPAA with 5-CQA significantly reduced PPAA activity in a dose dependent manner with an IC50 value of about 2mgmL(-1). Lineweaver-Burk plots indicated that 5-CQA exerts a mixed type inhibition as km increased and Vmax decreased. The total polyphenol content (TPC) of peeled tuber tissue ranged from 320.59 to 528.94mg 100g(-1)dry weight (DW) in raw tubers and 282.03-543.96mg 100g(-1)DW in cooked tubers. With the exception of Désirée, TPC and 5-CQA levels decreased after cooking. Principle component analysis indicated that digestibility is affected by multiple factors including phenolic, dry matter and starch content.

Aqueous extract of Psidium guajava leaves: phenolic compounds and inhibitory potential on digestive enzymes

ABSTRACT Leaves of Psidium guajava L. (guava) have been widely used in the popular way for prevention and treatment of various diseases. Thus, the objective of this study was to evaluate the inhibitory potential of leaves aqueous extract from three cultivars of P. guajava (Pedro Sato, Paluma and Século XXI) on α-amylase, α-glycosidase, lipase, and trypsin enzymes, in the presence or not of simulated gastric fluid and to determine the content of phenolic compounds by high performance liquid chromatography. All cultivars presented the same composition in phenolic compounds, but in different proportions. The compounds identified are gallic acid, epigallocatechin gallate, syringic acid, o-coumaric acid, resveratrol, quercetin, and catechin (which was the major compound in all the cultivars evaluated). In the absence of simulated gastric fluid, it was observed different inhibitions exercised by the leaves aqueous extracts from three cultivars of P. guajava on each enzyme. In presence of simulated gastric fluid, all cultivars showed increase in the inhibition of lipase and α-glycosidase, and decrease in inhibition of α-amylase and trypsin enzymes. These results indicate that P. guajava leaves aqueous extracts from all cultivars evaluated possess potential of use as an adjuvant in the treatment of obesity and other dyslipidemias.

Characterization of Lactobacillus salivarius strains B37 and B60 capable of inhibiting IL-8 production in Helicobacter pylori-stimulated gastric epithelial cells.

BACKGROUND: Interleukin (IL)-8 is the key agent for initiating an inflammatory response to infection with Helicobacter pylori. Some strains of Lactobacillus spp. are known to colonize the stomach and suppress inflammation caused by H. pylori. In this study, we characterized two gastric-derived lactobacilli, Lactobacillus salivarius (LS) strains B37 and B60, capable of inhibiting H. pylori-induced IL-8 production by gastric epithelial cells. RESULTS: Conditioned media from LS-B37 and LS-B60 suppressed H. pylori-induced IL-8 production and mRNA expression from AGS cells without inhibiting H. pylori growth. These conditioned media suppressed the activation of NF-κB but did not suppress c-Jun activation. IL-8 inhibitory substances in conditioned media of LS-B37 and LS-B60 are heat-stable and larger than 100 kDa in size. The inhibitory activity of LS-B37 was abolished when the conditioned medium was treated with α-amylase but still remained when treated with either proteinase K, trypsin, lipase or lysozyme. The activity of LS-B60 was abolished when the conditioned medium was treated with either amylase or proteinase K but still remained when treated with lysozyme. Treatment with lipase and trypsin also significantly affected the inhibitory activity of LS-B60 although the conditioned medium retained IL-8 suppression statistically different from media control. CONCLUSIONS: These results suggest that L. salivarius strains B37 and B60 produce different immunomodulatory factors capable of suppressing H. pylori-induced IL-8 production from gastric epithelial cells. Our results suggest that the large, heat-stable immunomodulatory substance(s) present in the LCM of LS-B37 is a polysaccharide, while the one(s) of LS-B60 is either complex consisting of components of polysaccharide, lipid and protein or includes multiple components such as glycoprotein and lipoprotein.

In vitro bioaccessibility and functional properties of polyphenols from pomegranate peels and pomegranate peels-enriched cookies.

Obesity is an urgent social problem and new functional foods providing polyphenols and dietary fibers (DF) may be promising tools to modulate oxidative stress, inflammation and energy homeostasis. Pomegranate peels (PPe) are an agro-industrial by-product containing polyphenols such as ellagitannins (ETs), gallic acid (GA), ellagic acid (EA) and its derivatives (EAs), as well as DF. In this study, PPe enriched cookies (PPeC) were developed, and the bioaccessibility as well as the ability of their polyphenols to exert antioxidant activity along the Gastro-intestinal Tract (GiT) and to modulate digestive enzymes were evaluated in vitro. Data showed that the potential bioaccessibility of ETs was 40% lower from PPeC than PPe whereas EAs' and GA bioaccessibility increased by 93% and 52% for PPeC compared to PPe. The concentration of the polyphenols at each digestion step was associated with the total antioxidant capacity of the potentially bioaccessible material. Moreover the polyphenols released in the simulated duodenal phase upon PPeC digestion exhibited inhibitory activity towards α-glucosidase, α-amylase and lipase, being α-glucosidase > α-amylase > lipase. In conclusion, the data demonstrated that the inclusion of PPe at 7.5% in a bakery product potentially led to a high bioaccessibility of ETs' degradation products (mainly EA and EAs) in the duodenum, with a consequent antioxidant protection along the GiT and modulation of glucose metabolism. Further human studies are warranted to evaluate whether these effects also occur in vivo.

Attenuation of Porphyromonas gingivalis oral infection by α-amylase and pentamidine.

The Porphyromonas gingivalis bacterium is one of the most influential pathogens in oral infections. In the current study, the antimicrobial activity of α-amylase and pentamidine against Porphyromonas gingivalis was evaluated. Their in vitro inhibitory activity was investigated with the agar overlay technique, and the minimal inhibitory and bactericidal concentrations were determined. Using the bactericidal concentration, the antimicrobial actions of the inhibitors were investigated. In the present study, multiple techniques were utilized, including scanning electron microscopy (SEM), general structural analysis and differential gene expression analysis. The results obtained from SEM and bactericidal analysis indicated a notable observation; the pentamidine and α-amylase treatment destroyed the structure of the bacterial cell membranes, which led to cell death. These results were used to further explore these inhibitors and the mechanisms by which they act. Downregulated expression levels were observed for a number of genes coding for hemagglutinins and gingipains, and various genes involved in hemin uptake, chromosome replication and energy production. However, the expression levels of genes associated with iron storage and oxidative stress were upregulated by α-amylase and pentamidine. A greater effect was noted in response to pentamidine treatment. The results of the present study demonstrate promising therapeutic potential for α-amylases and pentamidine. These molecules have the potential to be used to develop novel drugs and broaden the availability of pharmacological tools for the attenuation of oral infections caused by Porphyromonas gingivalis.

Alpha-amylase is a human salivary protein with affinity to lipopolysaccharide of Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans lipopolysaccharide (Aa.LPS) is a major virulence factor associated with aggressive periodontitis. Although the recognition of Aa.LPS is potentially initiated by salivary proteins in the oral cavity, Aa.LPS-binding proteins (Aa.LPS-BPs) in saliva are poorly characterized. The purpose of this study was to capture and identify Aa.LPS-BPs in human saliva using a LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Aa.LPS conjugated onto N-hydroxysuccinimidyl-Sepharose(®) 4 Fast Flow beads (Aa.LPS-beads) activated Toll-like receptor 4 and produced nitric oxide and Interferon gamma-inducible protein-10, implying that the conjugation process did not alter the biological properties of Aa.LPS. Aa.LPS-BPs were subsequently isolated from the nine human saliva samples from healthy individuals with the Aa.LPS-beads followed by identification with the mass spectrometry. Aa.LPS-BPs include α-amylase, serum albumin, cystatin, lysozyme C, submaxillary gland androgen-regulated protein 3B, immunoglobulin subunits, polymeric immunoglobulin receptor, deleted in malignant brain tumors 1, prolactin-inducible protein, lipocalin-1, and basic salivary proline-rich protein 2. Specific binding was validated using a pull-down assay with α-amylase which was captured at the highest frequency. Alpha-amylase demonstrated to interfere with the adherence and biofilm formation of A. actinomycetemcomitans. Even heat-inactivated α-amylase showed the interference to the same extent. Conclusively, we identified unique Aa.LPS-BPs that provide useful information to understand bacterial pathogenesis and host innate immunity in the oral cavity.

In vitro real-time interactions of cranberry constituents with immobilized fructosyltransferase.

Cranberry has been proposed as an anti-biofilm agent that does not kill bacteria, but rather prevents the pathogen from survival in the host. This can be achieved by inhibiting the function of virulent factors essential for the pathogen to persist in a host environment. The oral bacterial enzyme fructosyltransferase (FTF) plays an important role in the pathogenesis of dental diseases. The real-time interaction of cranberry nondialyzable material (NDM) with immobilized FTF was investigated using the surface plasmon resonance (SPR) technique. To determine its binding efficiency, NDM at concentrations between 0 µg/mL and 200 µg/mL was applied onto the immobilized FTF. The effect of NDM or other polyphenols, myricetin, and epicatechin on FTF enzymatic activity was evaluated by applying the above compounds and sucrose onto immobilized FTF. Salivary amylase was applied with NDM onto immobilized FTF to explore the effect on NDM-FTF interaction. Our results show that NDM firmly attaches to immobilized FTF in a dose-dependent manner, whereas the presence of salivary amylase reduced this binding interaction. Using nonlinear regression we calculated that the affinity constant of NDM applied alone (106 M⁻¹) was fivefold higher than NDM in the presence of amylase (0.2 x 106 M⁻¹). At 200 µg/mL, NDM, introduced together with sucrose, inhibited the activity of immobilized FTF by 63% within minutes, in comparison with the control (sucrose alone). The effect of NDM was sustained even after it was washed off the immobilized FTF. Myricetin also strongly inhibited FTF activity, whereas epicatechin was less effective. The real-time SPR observation suggests that one of the anti-biofilm modes of action of NDM is an immediate and irreversible inhibitory effect on the activity of immobilized FTF, which is due to a strong binding affinity to the immobilized enzyme.

Combination of enzymes and flow perfusion conditions improves osteogenic differentiation of bone marrow stromal cells cultured upon starch/poly(epsilon-caprolactone) fiber meshes.

Previous studies have shown that alpha-amylase and lipase are capable of enhancing the degradation of fiber meshes blends of starch and poly(epsilon-caprolactone) (SPCL) under dynamic conditions, and consequently to promote the proliferation and osteogenic differentiation of bone marrow stromal cells (MSCs). This study investigated the effect of flow perfusion bioreactor culture in combination with enzymes on the osteogenic differentiation of MSCs. SPCL fiber meshes were seeded with MSCs and cultured with osteogenic medium supplemented with alpha-amylase, lipase, or a combination of the two for 8 or 16 days using static or flow conditions. Lipase and its combination with alpha-amylase enhanced cell proliferation after 16 days. In addition, the flow perfusion culture enhanced the infiltration of cells and facilitated greater distribution of extracellular matrix (ECM) throughout the scaffolds in the presence/absence of enzymes. A significant amount of calcium was detected after 16 days in all groups cultured in flow conditions compared with static cultures. Nevertheless, when alpha-amylase and lipase were included in the flow perfusion cultures, the calcium content was 379 +/- 30 microg/scaffold after as few as 8 days. The highest calcium content (1271 +/- 32 microg/scaffold) was obtained for SPCL/cell constructs cultured for 16 days in the presence of lipase and flow. Furthermore, von Kossa staining and tetracycline fluorescence of histological sections demonstrated mineral deposition within the scaffolds for all groups cultured for 16 days under flow. However, all the data corroborate that lipase coupled with flow perfusion conditions improve the osteogenic differentiation of MSCs and enhance ECM mineralization.

Influência do fumo na atividade da amilase salivar e na curva glicêmica / Influence of smoking on salivary amylase activity and glycemic curve

OBJETIVO: Determinar a atividade da amilase salivar e a relação com a glicemia, antes e após a ingestão de carboidratos em fumantes e não fumantes, uma vez que in vitro a exposição da saliva à fumaça do cigarro induz à redução da atividade da amilase salivar e poderia influenciar na absorção dos carboidratos da dieta. MÉTODOS: Foram avaliados voluntários fumantes (n=10) e não fumantes (n=10). Realizou-se coletas da saliva antes e após o fumo e determinou-se a glicemia antes e após a ingestão de 72g de carboidratos. Para glicemia usaram-se tempos de 0, 15, 30, 60, 120 minutos. A determinação da atividade da amilase salivar foi realizada por meio de kits comerciais. A glicemia foi determinada utilizando o aparelho Glicomiter (Accu-Chek-Roche). As análises estatísticas foram realizadas no software Sigmastat, utilizou-se o método Teste t pareado (p<0,05). RESULTADOS: O aumento da glicemia aos 15, 30, 60 e 90 minutos foi de 3,9; 11,9; 34,8 e 22,7 por cento para os não fumantes e 4,9; 6,5; 13,8 e 9,7 por cento para os fumantes, respectivamente. No pico máximo de absorção tem-se uma glicemia de 21,0 por cento maior nos pacientes não fumantes. A atividade da amilase salivar antes e após alimentação apresentou-se 75,0 por cento menor nos indivíduos fumantes. CONCLUSÃO: Estes resultados sugerem que o fumo inibe a amilase e influencia na diminuição da digestão/absorção de carboidratos, consequentemente na concentração de glicose sanguínea, reduzindo assim o aporte energético ingerido.

OBJECTIVE: The objective of this study was to determine salivary amylase activity and its relationship with glycemia before and after smokers and nonsmokers ingested carbohydrates. Since cigarette smoke reduces salivary amylase activity in vitro, it may affect dietary carbohydrate absorption. METHODS: Twenty volunteers participated in this study, 10 smokers and 10 nonsmokers. Samples of saliva were collected before and after the smokers had a cigarette and glycemia was determined before and after the ingestion of 72g of carbohydrates. Glycemia was measured 0, 15, 30, 60 and 120 minutes after carbohydrate intake. Salivary amylase activity was determined by commercial kits. Glycemia was determined by a glucometer (Accu-Chek-Roche). The paired t-test was used for the statistical analyses, done by the software Sigmastat, with p<0.05. RESULTS: Glycemia 15, 30, 60 and 90 minutes after carbohydrate intake rose 3.9 percent, 11.9 percent, 34.8 percent and 22.7 percent in nonsmokers and 4.9 percent, 6.5 percent, 13.8 percent and 9.7 percent in smokers, respectively. The peak glucose absorption in nonsmokers was 21.0 percent greater than in smokers. Salivary amylase activity before and after eating was 75.0 percent smaller in smokers. CONCLUSION: These results suggest that smoking inhibits amylase and has a negative impact on the digestion/absorption of carbohydrates, consequently in blood glucose levels, thereby reducing the amount of energy absorbed.

Inhibitory effect of the lectin wheat germ agglutinin (WGA) on the proliferation of AR42J cells.

The rat pancreatic acinar tumour cell line AR42J is a widely used model to study the secretion, proliferation and differentiation of cells under the influence of hormones. These so-called amphicrine cells synthesize and secrete digestive enzymes as well as neuroendocrine peptides. They possess both subtypes of the highly glycosylated cholecystokinin (CCK) receptor which are important for the regulation of secretion and for cell growth. AR42J cells extrude CCK and gastrin-like hormone peptides and have the ability of an autostimulation (autocrine loop). The lectins wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I) bind to the glycosylated sites of these CCK receptors with the effect inhibiting CCK binding and thus inhibiting the CCK-induced Ca2+ release and alpha-amylase secretion. The so-called trophic hormones CCK and gastrin stimulate the secretion and proliferation of AR42J cells within the autocrine loop via autostimulation of their CCK receptors. In preceding papers, we described the inhibitory effect of WGA on the binding of 125I-CCK-8s to the CCK-A and -B receptors and the subsequent enzyme secretion of AR42J cells. In the present work, we studied the influence of the lectins WGA, UEA-I and galectin-1, as well as of the lectin-like enzyme alpha-amylase, on the proliferation of AR42J cells and prevention of autostimulation. The proliferation inhibition of the growth fraction was measured by estimation of the S-phase fraction by DNA flow cytometry. Whereas WGA inhibited the growth fraction significantly, UEA-I, human galectin-1 and human alpha-amylase had no significant effect. In transmission electron microscopy, we observed the accumulation of typical zymogen granules under the effect of WGA and a better differentiation of cells.

Effect of enzymes on the growth of human and animal rotaviruses.

The growth of group A human, bovine, equine and porcine rotaviruses were enhanced by pretreatment of virus with pancreatin, trypsin, protease, alkaline phosphatase or pepsin and incorporation of these enzymes in maintenance medium. In contrast, alpha-amylase or lipase inhibited the growth of equine and porcine rotaviruses. The other enzymes, adenosine deaminase, lactase, lysozyme, ribonuclease or triose-phosphate isomerase gave little or no change in the growth of all four rotaviruses.

Genital tract growth factors from a moth, the tobacco budworm, Heliothis virescens.

Development and maturation of the genital tract of the moth, Heliothis virescens, takes place within a few days in the pupal stage. The insect steroid hormone, 20-hydroxyecdysone (20E), essential during this period, stimulated testis sheath and fat body tissue to secrete factors that, in turn, stimulated growth and development of pupal spermducts and genital imaginal discs in vitro. Factors could be extracted in aqueous solution from tissues incubated for 24 h in media containing 1000pg/microliters 20E, and partially purified by chromatography on polyacrylamide gels. Ten active molecular weight fractions were separated from testis sheath extracts, and 9 from fat body extracts. Most fractions were labile to protease, although the activity of six of the fractions was also destroyed by lipase. Testis sheath, fat body tissue, and active fractions, caused partial development of the genital tract in vitro, as well as increased incorporation of [3H]methionine into precipitable protein and [3H]thymidine into nuclear material.

Effect of salivary proteins on binding curves of three radioimmunoassay kits: Amerlex-M progesterone, Amerlex cortisol, and Biodata testosterone.

Radioimmunoassay (RIA) is generally used to measure certain salivary hormones because of its high sensitivity. For speed and simplicity, it has been used in the form of "direct" assays, i.e., without first extracting the analyte from its matrix. Investigating the effect of the principal salivary proteins on the binding behavior of three commercial RIA kits, we found that the Amerlex-M [125I]progesterone binding was greatly reduced when alpha-amylase and mucins were added to the binding medium, whereas IgA and IgG were less effective. The Serono Biodata [125I]testosterone binding was unaffected by proteins, while the Amerlex [125I]cortisol binding was decreased by alpha-amylase and mucins. The protein influence was largely eliminated when an extraction step was incorporated. Thus, direct RIA of saliva may be subject to matrix effects, to extents that vary with the kit and that may adversely affect the quality of the assay results.

Comparison of gamma-glutamyl hydrolase (conjugase; EC 3.4.22.12) and amylase treatment procedures in the microbiological assay for food folates.

1. It has been suggested that deconjugation of food folates with pig kidney, compared with chicken pancreas, may increase measureable folate by approximately 50% (Phillips & Wright, 1983). Therefore deconjugation with conjugases from these two enzyme sources was optimized and compared. Folate was measured microbiologically with Lactobacillus casei (ATCC 7469) as the test organism at pH 5.6. 2. Treatment for 6 h with 200 mg pig kidney/200 mg sample was compared with the conventional assay employing overnight incubation with 20 mg chicken pancreas/5 g sample. Comparison of the deconjugation systems showed chicken pancreas to be superior for peas (Pisum sativum) and beans (Phaseolus vulgaris), while there was no difference for potatoes. 3. gamma-Glutamyl hydrolase (conjugase; EC 3.4.22.12) treatment for 2 and 20 h with pig kidney and chicken pancreas was optimized for raw potatoes and green frozen peas. Treatments with pig kidney were conducted at pH 4.6, which is optimal, and at pH 5.2. There was no significant difference between 2 and 20 h treatments at pH 5.2. Treatments with chicken pancreas were conducted at pH 6.1. Treatment for 2 h was preferred as it resulted in significantly higher measureable folate activity in peas and potatoes, and because overnight treatment can be influenced by microbial production of folate. 4. With optimal treatment conditions the source of enzymes did not significantly influence measureable folate activity. Chicken pancreas is the traditional source of conjugase in Scandinavia and was preferred for deconjugation. 5. Chicken pancreas, 20 and 60 mg, was used for deconjugation of sixteen different food samples, each containing approximately 200 ng folate. Chicken pancreas at 60 mg/sample gave significantly higher results. Further addition of enzyme did not increase the folate values. 6. A combined amylase treatment using heat-resistant alpha-amylase (EC 3.2.1.1) during extraction to ensure the gelatinization of the sample, and glucan 1,4-alpha-glucosidase (amyloglucosidase; EC 3.2.1.3), along with the incubation with chicken pancreas to complete the digestion, was shown to give a small but significant increase in folate values of starch-containing samples. 7. Folate results using the recommended procedure are given for raw potatoes, wheat bran, rolled oats, wheat flour and dark rye bread. 8. Chicken pancreas was shown to contain equally high amounts of amylases as did the alpha-amylase and amyloglucosidase sources. This finding may explain the relatively small benefit of a specific amylase addition.

Pharmacodynamics and tolerability of low doses of tendamistate given with starch.

Six healthy male volunteers participated in a double-blind cross-over study in which 12,5 mg, 25 mg, or 50 mg tendamistate (HOE 467), an alpha-amylase inactivator, or placebo were administered with 100 g maize meal, which contains 85,5 g pure starch. Serial blood specimens for measurement of plasma glucose concentrations, which served as a criterion of starch absorption, were taken up to 3 hours after each starch meal, and side-effects were recorded. The administration of HOE 467 with a starch meal resulted in a significant inhibition of starch absorption. Side-effects were not severe. Flatulence occurred in the placebo phase as well as after administration of the active drug. Loose stools were reported by 1 subject after 25 mg tendamistate.