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.

Research Note: Corn energy and nutrient utilization by broilers as affected by geographic areas and carbohydrases.

Two experiments (Exp.) were conducted to evaluate the effects of exogenous carbohydrases on nutrient and energy utilization of corn with different compositions by broilers. In Exp. 1, a total of 448 Cobb 500 male chicks were distributed in a 2 × 4 factorial arrangement (corn from regions geographically located in the North or South of Brazil × 4 carbohydrases supplementation), with 8 replicate cages of 7 birds each. In Exp. 2, 672 Cobb 500 male chicks were fed 12 experimental feeds, in a 3 × 4 factorial arrangement [3 corn endosperm compositions (waxy, semi-dent, or semi-flint) × 4 carbohydrases], with 8 replicate cages of 7 birds. Birds were fed semi-purified test diets with 95.9% corn from d 14 to 24 in both studies. In Exp. 1, α-amylase, ß-xylanase, or carbohydrase complex (cellulase, glucanase, and xylanase) were added to the diet. In Exp. 2, α-amylase, ß-xylanase, or α-amylase + ß-xylanase were supplemented. Digestibility of DM, N, ether extract (EE), Ca, and P as well as AME, AMEn, and IDE were determined. In Exp. 2, jejunal starch digestibility was determined on d 24. Data were subjected to analysis of variance and means were compared by Tukey test (P ≤ 0.05). Corn from the North origin had higher AME, AMEn, and digestibility of DM and N compared to the South (P ≤ 0.05). Amylase supplementation led to increases of 3% in AME and 2% in N digestibility when compared to the non-supplemented feeds (P ≤ 0.01). In Exp. 2, the highest ME values and EE digestibility were observed in the semi-flint corn compared to the waxy, whereas the semi-dent presented the highest digestibility of N and starch. Corn diets supplemented with amylase + xylanase had improvements of 2.5% AMEn and 3% starch digestibility. In conclusion, energy and nutrient utilization of corn by broilers depend on the region where it was grown. Corn genetics, expressed by the endosperm composition, and carbohydrase supplementation influenced energy and nutrient utilization by broilers.

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.

Incredible affinity of Kattosh with PPAR-γ receptors attenuates STZ-induced pancreas and kidney lesions evidenced in chemicobiological interactions.

Since ancient times, plants have been used as green bioresources to ensure a healthier life by recovering from different diseases. Kattosh (Lasia spinosa L. Thwaites) is a local plant with various traditional uses, especially for arthritis, constipation and coughs. This research investigated the effect of Kattosh stem extract (LSES) on streptozotocin-induced damage to the pancreas, kidney, and liver using in vitro, in vivo and in silico methods. In vitro phytochemical, antioxidative and anti-inflammatory effects of LSES were accomplished by established methods followed by antidiabetic actions in in vivo randomized controlled intervention in STZ-induced animal models for four weeks. In an in silico study, LSES phytocompounds interacted with antidiabetic receptors of peroxisome proliferator-activated receptor-gamma (PPAR, PDB ID: 3G9E), AMP-activated protein kinase (AMPK, PDB ID: 4CFH) and α-amylase enzyme (PDB ID: 1PPI) to verify the in vivo results. In addition, LSES showed promising in vitro antioxidative and anti-inflammatory effects. In contrast, it showed a decrease in weekly blood glucose level, normalized lipid profile, ameliorated liver and cardiac markers, managed serum AST and ALT levels, and increased glucose tolerance ability in the animal model study. Restoration of pancreatic and kidney damage was reflected by improving histopathological images. In ligand-receptor interaction, ethyl α-d-glucopyranoside of Kattosh showed the highest affinity for the α-amylase enzyme, PPAR, and AMPK receptors. Results demonstrate that the affinity of Kattosh phytocompounds potentially attenuates pancreatic and kidney lesions and could be approached as an alternative antidiabetic source with further clarification.

Screening of antimicrobial, antioxidant, antidiabetic activities, anatomical and morphological properties of Colchicum speciosum Steven (Colchicaceae).

Colchicum speciosum Steven species is a perennial stemless plant. C. speciosum is a flowering herb native to mountainous regions of northern Turkey, the Caucasus, and northern Iran. It has been known as "Vargit, Aci Çigdem, Güz Çigdemi". The present study reports the antimicrobial, antioxidant, α-amylase and α-glucosidase inhibitory activities of corm, leaf and flower methanol extracts, anatomical (light and electron microscopes) properties of root, corm, leaf, flowers and morphological characteristics of C. speciosum. Three different part of extracts C. speciosum were evaluated against E. coli ATCC 8739, S. aureus ATCC 6538, B. subtilis ATCC 19,659, C. albicans ATCC 10,231, C. krusei ATCC 14,243, and C. tropicalis ATCC 750. The most effective extract was found to be MeOH extract for corm and leaf against C. tropicalis ATCC 750 strain with MIC value 160 > µg/mL. It has been investigated first time anatomy of the tepal, ovary, anther, filament of C. speciosum. Leaf extract was the highest phenolic component (78.61842 µg GAE/ mg extract). As a result of DPPH• and ABTS•+ tests, it was determined that the leaf extract showed the best activity (IC50 = 6.568 µg/mL and IC50 = 3.243 µg/mL, respectively). Corm extract exhibited α-glucosidase inhibitory activity with an IC50 value of 21039 µg/mL. This is the first study of the in vitro antimicrobial, antioxidant, antidiabetic activities, detailed anatomical and morphological properties of C. speciosum. HiGHLiGHTS : • Antioxidant-antidiabetic-antimicrobial potential of Colchicum speciosum • Leaf extract had the highest phenolic component • The leaf got the highest DPPH• and ABTS•+ antioxidant potential • Corm extract exhibited α-glucosidase inhibitory activity • The most effective extract was found to be MeOH extract for corm and leaf against C. tropicalis • This is the first study of the in vitro antimicrobial, antioxidant, antidiabetic activities, detailed anatomical and morphological properties of C. speciosum.

Impact of feed supplement with alfa-amylase and beta-glucanase on ingestive-related biomarkers registered with real-time sensors.

The aim of this study was to investigate the impact of feed supplements with alfa-amylase and beta-glucanase (Optipartum C+ 200) on ingestive-related behaviour biomarkers registered with real-time sensors: rumination behaviours and reticulorumen parameters (pH and temperature). Cows (n=20) in the treatment group (TG) were fed with Optipartum C+ 200 (Enzymes feed supplement: Alfa-Amylase 57 Units; Beta-Glucanase 107 Units) from 21 days before calving until 30 days after calving with a feeding rate of 200 g/cow/day. Cows (n=22) in the control group (CG) were fed a feed ration without feed supplement. Measurements started from 6 days before calving and continued until 21 days after calving. The following indicators were registered: with the RumiWatch System: Rumination time; Eating time; Drinking time; Rumination chews; Eating chews; Drinking gulps; Bolus; Chews per minute; Chews per bolus. With the SmaXtec system: the temperature, pH of the contents of the cows' reticulorumens, and cows' walking activity. According to our results, feed supplementation with alfa-amylase and beta-glucanase (Optipartum C+ 200) in the TG group resulted in increases in the following parameters: 9% rumination time and eating time, 19% drinking time, 11% rumination chews, 16% eating chews, 13% number of boluses per rumination, 5% chews per minute and 16% chews per bolus. The rumination time showed a strong, positive relation with rumination chews and bolus indicators in both groups (TG and CG) (p⟨0.001); while the rumination time in both groups of cows showed an opposite direction and was negatively related to eating time and eating chews (p⟨0.05). We found a 1.28 % lower reticulorumen pH and a 0.64 % lower reticulorumen temperature in cows fed with the supplement compared with cows in the control group. Cows in TG were 8.80% more active than those in the CG group. For improvement of ingestive-related behaviour we suggest adding a feed supplement with alfa-amylase and beta-glucanase (Optipartum C+ 200).

Growth phase and dietary α-amylase supplementation effects on nutrient digestibility and feedback enzyme secretion in broiler chickens.

Growth performance, nutrient digestibility, intestinal health, and endogenous enzyme secretion responses to dietary α-amylase supplementation during 4 growth phases of broiler chickens fed corn-soybean meal-based diets were evaluated in the present study. A total of 1,136 male broiler chicks were assigned at day 0 after hatching to 8 treatments in a 2 × 4 factorial arrangement. There were 2 dietary levels of α-amylase supplementation of 0 or 80 kilo-Novo alpha amylase units per kg diet and 4 posthatching growth phases of day 0 to 11, day 11 to 21, day 21 to 42, or day 42 to 56 in a randomized complete block design. Each treatment comprised 8 replicate pens, with either 25 (day 0-11), 20 (day 11-21), 16 (day 21-42), or 10 (day 42-56) birds per pen. Body weight gain and feed efficiency of birds improved (P < 0.01) with α-amylase supplementation. There were main effects of α-amylase, growth phase, and interaction (P < 0.01) on apparent ileal digestibility (AID) of starch. This ranged from 0.8% during day 11 to 21 to 2.8% during day 0 to 11 after hatching. The total tract retention of starch increased (P < 0.05) with amylase supplementation but was not different across growth phases. Amylase supplementation increased (P < 0.05) AID of gross energy, AME (kcal/kg), and AMEn (kcal/kg). Villus height in the jejunal tissue was increased (P < 0.01) by α-amylase supplementation. During day 11 to 21 after hatching, the viscosity of jejunal digesta and pancreatic amylase activity increased (P < 0.01) with amylase supplementation. In conclusion, dietary amylase supplementation improved growth performance, apparent nutrient digestibility, and digestive enzyme activity of broiler chickens fed a corn-soybean diet. The study indicates that the growth phase of birds may affect response to exogenous amylase.

Intestinal starch and energy digestibility in broiler chickens fed diets supplemented with α-amylase.

Dietary starch is the major energy source for broiler chickens; therefore, relevant information on its intestinal utilization is important. The present study was designed to evaluate intestinal starch and energy digestibility of broiler chickens fed diets supplemented with α-amylase. A total of 240 day-0 male broiler chicks were randomly assigned to 3 nutritionally adequate corn-soybean-based experimental diets comprising 3 levels of α-amylase supplementation (0, 80, or 160 KNU/kg diet). Each treatment comprised 8 replicate cages of 10 birds each. At day 21 after hatching, digesta was collected from 4 intestinal sites: the anterior jejunum (AJ), posterior jejunum (PJ), anterior ileum (AI), and posterior ileum. Increasing α-amylase supplementation linearly improved (P < 0.01) overall BW gain and feed efficiency of the birds. There were linear and quadratic (P < 0.01) responses of increasing α-amylase supplementation on starch and energy digestibility at the PJ and AI. The total tract digestibility of starch increased (P < 0.05) with increasing α-amylase supplementation. Starch disappearance and digestible energy (kcal/kg) linearly increased (P < 0.01) with digesta flow from the AJ to PJ as dietary α-amylase supplementation increased. There were linear (P < 0.01) and quadratic (P < 0.05) effects of increasing α-amylase supplementation on the villus height in the jejunum. The viscosity of the jejunal digesta decreased (P < 0.05) with increasing dietary α-amylase supplementation. The results from this study showed the efficacy of exogenous amylase in improving growth performance and starch and energy digestibility in broiler chickens. Furthermore, the digestibility of starch and energy and the impact of the exogenous amylase were higher at the PJ than other intestinal sites.

Medicinal properties of Ocotea bullata stem bark extracts: phytochemical constituents, antioxidant and anti-inflammatory activity, cytotoxicity and inhibition of carbohydrate-metabolizing enzymes.

OBJECTIVE: This study evaluates the phytochemical constituents, antioxidant and anti-inflammatory activity, cytotoxicity, and inhibitory activity against carbohydrate metabolism of extracts from Ocotea bullata stem bark. METHODS: Hexane, ethyl acetate, methanol and water were used to extract the air-dried sample. The phytochemical investigation and antioxidant assays were carried out on the extracts using standard procedures. The antidiabetic and anti-inflammatory potentials were evaluated using α-amylase, α-glucosidase and 5-lipoxygenase enzymes respectively. Vero cells were employed to determine the cytotoxicity of the extracts. RESULTS: The ethyl acetate extract showed higher phenolic contents (8.97 mg/g gallic acid) while methanol displayed higher flavonoid (36.06 mg/g quercetin) and flavonol (153.44 mg/g rutin) contents than other extracts. Hexane extract had the greatest capacity to scavenge 1,1-diphenyl-2-picryl-hydrazyl (0.19 mg/mL), hydroxyl (25.77 mg/mL) and 2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (0.07 mg/mL) radicals, while ethyl acetate extract exhibited stronger inhibition (P < 0.05) against superoxide anion (0.41 mg/mL) and ferric ion-reducing power (2.36 mg/mL) compared to other extracts and standards. Aqueous extract (27.02 mg/mL) exhibited strong metal-chelating activity (P < 0.05) compared to other extracts and gallic acid. The aqueous extract demonstrated the greatest inhibition of α-glucosidase (1.45 mg/mL) and α-amylase (2.43 mg/mL) compared to other extracts and acarbose. There were no significant differences (P < 0.05) in half-maximum inhibitory concentration (IC50) values of all tested extracts and indomethacin in the inhibition of 5-lipoxygenase activity. The aqueous extract was nontoxic to Vero cells with an IC50 value of 0.38 mg/mL. CONCLUSION: O. bullata stem bark contains active phytochemicals with diverse pharmacological potentials that could be beneficial in managing diabetes and inflammation.

THESIS-ABSTRACT Supplementation levels of exogenous alpha-amylase in broilers diets.

This study aimed to evaluate the supplementation levels of an exogenous alpha-amylase in broilers diets and compare two indicators in determining the diets energy. The experiment was divided into two parallel evaluations, being one of performance and the other of metabolism. In performance assay, 1,700 one-day-old Cobb-500 male chicks were used. The animals were distributed in 50 experimental plots and evaluated five treatments with ten replicates in a completely randomized design (CRD). The treatments were: a positive control (PC), a negative control (NC) and three alpha-amylase supplementation levels 200, 400 and 600 g/t, and the NC was formulated with 50 and 90 kcal of energy reduction in relation to the PC to the phases from 1 to 21 days and from 22 to 42 days, respectively. In the metabolism assay were used 240 animals, 150 birds for stage from 14 to 21 days and 90 birds to stage from 35 to 42 days of age and the treatments were the same as the performance assay, with six replicates per treatment in CRD. All diets of metabolism test contained the digestibility indicators Lipe® (eucalyptus purified lignin) and chromic oxide (Cr2O3), in concentrations of 0.05 and 1.0%, respectively. In the period from 1 to 21 days old, no significant differences were observed in weight gain (WG) (P > 0.05), however, feed intake (FI) was found higher by using 200 ppm of enzyme (P < 0.05) and better feed conversion (FC) with the PC (P < 0.05). From 22 to 42 days, no significant differences were observed on the WG (P > 0.05), but were observed lower FI and better FC to PC treatment (P < 0.05). In the period from 1 to 42 days of age, significant differences were also not observed on the WG (P > 0.05), but there was lower FI and better FC for the PC treatment (P < 0.05). The AMEn (apparent metabolizable energy corrected for nitrogen balance), determined using the total collection, reaffirmed the values ​​calculated for the PC and NC with intermediate data obtained from the enzyme use (200, 400 and 600 ppm). Comparing the total collection using Lipe® and Cr2O3, a correlation was observed only for the PC results, that were always higher, and for the NC results, that were lower for the three methodologies. For IDE (ileal digestible energy), determined by Cr2O3, significant differences were observed (P < 0.05) and it presented higher values in the PC treatment and lower values in the NC. In IDE determining by Lipe®, significant was observed (P < 0.05), showing higher value in the PC treatment. It is concluded that in the metabolism assessments, exogenous alpha-amylase tested was effective in increasing the metabolizable energy (ME), however, not enough to be equivalent to the ME of PC treatment, a result that was better expressed in the total collection, AMEn by Lipe® and IDE by Lipe® methodologies. The performance results reflect the superiority of PC treatment, pointing enzyme limitation to supply the energy deficit practiced in this work.

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

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.

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.

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.

Effect of Tea Catechins on Folate Analysis in Green Tea by Microbiological Assay.

Green tea is thought to be a primary source of folate in the Japanese diet, based on folate content analyzed by a microbiological assay. Green tea also contains high amount of catechins, in particular, epigallocatechin gallate (EGCg), which was demonstrated to be able to inhibit the digestive enzyme activities and microbial growth in the folate assay. In the present study, we examined whether tea catechins interfered with components of the folate assay for green tea. A marked inhibitory effect of EGCg on microbial growth was observed at an inhibitory concentration of higher than 10 µg/mL. Tea catechins without the galloyl moiety did not show an inhibitory effect. EGCg inhibited the activity of the three enzymes used for assay sample preparation at an inhibitory concentration of higher than 750 µg/mL for α-amylase, 1,000 µg/mL for protease, and 50 µg/mL for conjugase. However, with each step of the assay, the actual concentration of EGCg was decreased to below the inhibitory concentration of each analytical step. Lack of influence of EGCg on green tea folate assay was confirmed by an addition of folate standard in tea infusion. These results suggested that tea catechins have no practical impact on folate analysis in green tea, using the general microbiological assay.

Effect of α-Amylase, Papain, and Spermfluid treatments on viscosity and semen parameters of dromedary camel ejaculates.

Ejaculates from five clinically healthy dromedary camels (Camelus dromedarius) were used to evaluate the effects of different enzymatic treatments (Amylase, Papain, Spermfluid) on liquefaction and seminal parameters. After collection, ejaculates were divided into 5 aliquots: (1) kept undiluted (control); or diluted 1:1 with: (2) Tris-Citrate-Fructose (TCF), (3) TCF containing Amylase, (4) TCF containing Papain or (5) Spermfluid containing Bromelain. At 120 min after dilution, each aliquot was evaluated, at 20-min intervals, for viscosity, motility, viability and agglutination. Only the aliquots diluted with TCF containing Papain underwent complete liquefaction. Sperm motility decreased significantly during the observation times, except for the samples diluted with Spermfluid (P=0.005). Diluted samples showed different levels of agglutination, with the lowest being observed in the control and the highest in the Papain-treated samples. The viscosity of dromedary camel ejaculates could be effectively reduced by using the proteolytic enzyme Papain.

α-Amylase is a potential growth inhibitor of Porphyromonas gingivalis, a periodontal pathogenic bacterium.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis is a major etiological agent in the development and progression of periodontal diseases. In this study, we isolated a cell growth inhibitor against P. gingivalis species from rice protein extract. MATERIAL AND METHODS: The cell growth inhibitor active against P. gingivalis was purified from polished rice extract using a six-step column chromatography process. Its antimicrobial properties were investigated through microscope analysis, spectrum of activity and general structure. RESULTS: The inhibitor was identified as AmyI-1, an α-amylase, and showed significant cell growth inhibitory activity against P. gingivalis species. Scanning electron microscopy micrograph analysis and bactericidal assay indicated an intriguing possibility that the inhibitor compromises the cell membrane structure of the bacterial cells and leads to cell death. Moreover, α-amylases from human saliva and porcine pancreas showed inhibitory activity similar to that of AmyI-1. CONCLUSIONS: This is the first study to report that α-amylases cause cell death of periodontal pathogenic bacteria. This finding highlights the potential importance and therapeutic potential of α-amylases in treating periodontal diseases.

Role of phenolics as antioxidants, biomolecule protectors and as anti-diabetic factors--evaluation on bark and empty pods of Acacia auriculiformis.

OBJECTIVE: To search for an efficient and inexpensive source of phytoconstituents with antioxidant potential and health promoting traits from bark and empty pods of Acacia auriculiformis (A. auriculiformis). METHODS: Samples of bark and empty pod extracts were analyzed for bioactives (phenolics, flavonoids and proanthocyanidins) and subjected to free radical scavenging activity on DPPH˙, ABTS˙+, OH˙, O(2)•- and NO along with the determination of reducing power, iron chelating activity and peroxidation inhibition. Defensive action of extracts on biomolecules and cell membranes were evaluated by DNA nicking assay and haemolysis inhibition assay respectively. α-amylase and α-glucosidase inhibitory potentials were also determined. RESULTS: All the bioactives analyzed were higher in bark (B) than empty pods (EP) [TPC: B (574.51±16.11); EP (96.80±3.45) mg GAE/g. TFC: B (94.71±7.65); EP (247.87±20.45) mg RE/g. Proanthocyanidins: B (2.81±0.31); EP (1.25±0.01) mg LE/100 g DM] except flavonoids. Both the extracts showed higher quenching capacity on DPPH and ABTS (DPPH: B (0.21±0.01); EP (1.51±0.17) g extract/g DPPH. ABTS: B (111 519.14±79 340.91); EP (80 232.55±32 894.12) mmol TE/g) with the FRAP of B (84 515.63±3 350.69) and EP (47 940.79±1 257.60) mmol Fe (II)/g. Iron chelation was not observed. In addition, they showed lower quenching activity on OH(˙) (B (48.95±1.72); EP (34.94±1.62)%) and equivalent quenching on O(2)•- (B (53.47±3.92); EP (24.41±2.61)%), NO (B (49.04±5.04); EP (51.00±5.13)%), peroxidation inhibition (B (67.50±5.50); EP (55.1±2.3)%) and antihaemolytic potential (B (87.60±6.84)%) towards authentic antioxidant standards. Interestingly, Empty pod extracts are devoid of antihaemolytic activity. Both the extracts showed dose dependent DNA protection. Besides this, bark and empty pod extracts exhibited dual inhibiting potential against α -amylase and α-glucosidase enzymes. CONCLUSIONS: On summarization, it insinuated that both bark and empty pods can be used for the preparation of antioxidant/nutraceutical supplements and in anti-diabetic formulations.

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.