Amylolysis Kinetics Research Articles

Exploring antidiabetic potential of a polyherbal formulation Madhurakshak Activ: An in vitro and in silico study

Madhurakshak Activ (MA), a commercial polyherbal antidiabetic preparation is known to manage diabetes mellitus (DM) by reducing blood glucose levels. However, lacks systematic mechanistic evaluation for their molecular and cellular mode of actions. In the present study, hydro-alcoholic and aqueous extract of MA were evaluated for their effects on glucose adsorption, diffusion, amylolysis kinetics and transport across the yeast cells using in vitro techniques. Bioactive compounds identified from MA by LC-MS/MS were assessed for their binding potential against DPP-IV and PPARγ via an in silico approach. Our results revealed that the adsorption of glucose increased dose dependently (5 mM -100 mM). Both extracts exhibited linear glucose uptake into the yeast cells (5 mM - 25 mM), whereas glucose diffusion was directly proportional to time (30–180 min). Pharmacokinetic analysis revealed drug-like properties and low toxicity levels for all the selected compounds. Among the tested compounds, 6-hydroxyluteolin (−8.9 against DPP-IV and PPARγ) and glycyrrhetaldehyde (DPP-IV -9.7 and PPARγ −8.5) have exhibited higher binding affinity compared to the positive control. Therefore, the above compounds were further considered for molecular dynamics simulation which showed stability of the docked complexes. Hence, studied mode of actions might produce a concerted role of MA in increasing the rate of glucose absorption and uptake followed by the in silico studies which suggest that the compounds identified from MA may inhibit DPP-IV and PPARγ phosphorylation.

How Cooking Time Affects In Vitro Starch and Protein Digestibility of Whole Cooked Lentil Seeds versus Isolated Cotyledon Cells.

Lentils are sustainable sources of bioencapsulated macronutrients, meaning physical barriers hinder the permeation of digestive enzymes into cotyledon cells, slowing down macronutrient digestion. While lentils are typically consumed as cooked seeds, insights into the effect of cooking time on microstructural and related digestive properties are lacking. Therefore, the effect of cooking time (15, 30, or 60 min) on in vitro amylolysis and proteolysis kinetics of lentil seeds (CL) and an important microstructural fraction, i.e., cotyledon cells isolated thereof (ICC), were studied. For ICC, cooking time had no significant effect on amylolysis kinetics, while small but significant differences in proteolysis were observed (p < 0.05). In contrast, cooking time importantly affected the microstructure obtained upon the mechanical disintegration of whole lentils, resulting in significantly different digestion kinetics. Upon long cooking times (60 min), digestion kinetics approached those of ICC since mechanical disintegration yielded a high fraction of individual cotyledon cells (67 g/100 g dry matter). However, cooked lentils with a short cooking time (15 min) showed significantly slower amylolysis with a lower final extent (~30%), due to the presence of more cell clusters upon disintegration. In conclusion, cooking time can be used to obtain distinct microstructures and digestive functionalities with perspectives for household and industrial preparation.

Impact of Particle Size on the Rheological Properties and Amylolysis Kinetics of Ungelatinized Cassava Flour Suspensions

The effect of particle size on enzymatic hydrolysis of cassava flour at subgelatinization temperature was investigated. A multiscale physical metrology was developed to study the evolution of different physical-biochemical parameters: rheology, granulometry, and biochemistry. In this study, four fractions of cassava flour based on the particle sizes under 75 µm (CR075), 75–125 µm (CR125), 125–250 µm (CR250), and 250–500 µm (CR500) were screened for enzymatic hydrolysis effect. The results showed that all cassava flour suspensions exhibited a shear-thinning behavior, and the viscosity increased drastically with the increase of particle size. During hydrolysis, the viscosity reduced slightly and the non-Newtonian behavior became negligible beyond 4 h of the process. The particles size for CR075 and CR125 increased steadily in diameter mean. The samples of CR250 and CR500 showed more fluctuation by first decreasing, followed by increasing in particle sizes during the process. The highest hydrolysis yield was found for samples with particle size under 125 µm (89.5–90.7%), suggesting that mechanical treatment of cassava can enhance the bioconversion rate.

Revealing the mechanisms of starch amylolysis affected by tea catechins using surface plasmon resonance

The interaction of polyphenolic catechins from Camellia sinensis tea with α-amylase, and the effects of the interactions on the kinetics of starch amylolysis, were investigated. Binding studies using surface plasmon resonance (SPR) and enzyme kinetic analysis indicated that the in vitro digestibility of gelatinized wheat starch was decreased by the addition of catechins present in tea. Tea catechins decreased enzyme activity by reducing the maximum velocity (Vmax) of the α-amylase, but had little effect on the Michaelis-Menten constant (Km). Binding studies by SPR showed that the structure of the catechins influenced their affinity for α-amylase. Pearson correlation analysis showed that the decreased digestibility of starch in the presence of catechins was due to their binding of α-amylase interaction inhibiting the catalytic effectiveness of the enzyme. From this study, we concluded that the faster and stronger the binding of catechins and α-amylase, the greater reduction of starch digestion is.

Comparative in vitro hypoglycemic studies of unripe, ripe and overripe fruit extract of Musa paradisiaca (Indian banana)

PurposeIn the Indian system of medicine – Ayurveda, Musa paradisiaca has been mentioned as a remedy for various diseases and ailments. Based on the folkloric use, the purpose of this paper is to verify and compare the hypoglycemic potential of unripe, ripe and overripe fruit extract of Musa paradisiaca.Design/methodology/approachHypoglycemic activity of fruit extracts has been evaluated using various in vitro methods, namely, determination of glucose adsorption capacity, glucose uptake in yeast cells, amylolysis kinetics and glucose diffusion.FindingsThe extracts of unripe, ripe and overripe fruits of Musa paradisiaca adsorbed glucose, and the adsorption of glucose increased remarkably with an increase in glucose concentration. In the amylolysis kinetic experimental model, the rate of glucose diffusion was found to increase with time, and all the extracts of unripe, ripe and overripe fruits of Musa paradisiaca demonstrated significant inhibitory effects on the movement of glucose into external solution across the dialysis membrane as compared to the control. The extracts under study also promoted glucose uptake by the yeast cells in all the five glucose concentrations used in the study.Practical implicationsHere, the authors have verified and compared the hypoglycemic potential of Musa paradisiaca, its unripe fruit extract was found to show a better activity than ripe and overripe fruit extracts.Originality/valueBanana, being an all season readily available fruit, is widely consumed due to its ready availability and low cost. It acts as a complete food for even low socio-economic classes of society, owing to its rich nutritional values. Even in a processed and unprocessed manner, it is an important constituent of diet. The research suggests that instead of consuming ripe and overripe fruit, the unripe fruit will help in management of diabetes.

Hypoglycemic effects of Lagenaria siceraria, Cynodon dactylon and Stevia rebaudiana extracts

Introduction: The aim of the current analysis was to judge the hypoglycemic action of the phyto-extracts of Lagenaria siceraria, Cynodon dactylon and Stevia rebaudiana using suitable in vitro approaches. Methods: The hypoglycemic activity of the phyto-material extracts was evaluated by employing various in-vitro methods namely glucose diffusion, amylolysis kinetics and glucose adsorption capacity. Results: The extracts of L. siceraria, C. dactylon and S. rebaudiana exhibited glucose dialysis retardation indices (GDRI) of 48.14%, 37.03% and 29.62%, respectively at 60 minutes which were reduced to 15.78%, 10.52% and 18.42%, respectively at 120 minutes. All the plant extracts used in the study adsorbed glucose and their adsorptions markedly enhanced with increase in sugar concentration. Conclusion: From the outcome of the assay it can be concluded that the extracts of L. siceraria, C. dactylon and S. rebaudiana have hypoglycemic activity as observed in various in-vitro assays. However, the beneficial actions require to be verified by adopting various in vivo techniques along with clinical trials for their efficient use as potential remedial moiety.

In vitro hypoglycemic effects of unripe and ripe fruits of Musa sapientum

The present study was undertaken to verify the hypoglycemic potential of unripe and ripe fruit extracts of Musa sapientum by using various in-vitro techniques, namely glucose adsorption capacity, glucose diffusion, amylolysis kinetics and glucose transport across the yeast cells. The results revealed that the unripe and ripe fruit extracts of Musa sapientum adsorbed glucose and the adsorption of glucose increased remarkably with an increase in glucose concentration. There were no significant (p≤0.05) differences between their adsorption capacities. In the amylolysis kinetic experimental model the rate of glucose diffusion was found to be increased with time from 30 to 180 min and both extracts exhibited significant inhibitory effects on the movement of glucose into external solution across the dialysis membrane as compared to control. The plant extracts also promoted glucose uptake by the yeast cells and enhancement of glucose uptake was dependent on both the sample and glucose concentration. The hypoglycemic effect exhibited by the extracts was observed to be mediated by inhibiting α-amylase, inhibiting glucose diffusion by adsorbing glucose and by increasing glucose transport across the cell membranes as revealed by an in-vitro model of yeast cells.

IN VITRO HYPOGLYCEMIC EFFECTS OF CAESALPINIA BONDUCELLA AND MYRISTICA FRAGRANS SEED EXTRACTS

The present investigation was undertaken to assess the hypoglycemic potential of Caesalpinia bonducella (C.bonducella) and Myristica fragrans (M.fragrans), employing various in vitro techniques. The extracts of seeds of C. bonducella and M. fragrans were studied for their effects on glucose adsorption capacity, in vitro glucose diffusion, in vitro amylolysis kinetics and glucose transport across the yeast cells. It was observed that the plant extracts under study adsorbed glucose and the adsorption of glucose increased remarkably with an increase in glucose concentration. There were no significant (p≤0.05) differences between their adsorption capacities. The results of amylolysis kinetic experimental model revealed that the rate of glucose diffusion was found to be increased with time from 30 to 180 min and both the plant extracts demonstrated significant inhibitory effects on movement of glucose into external solution across dialysis membrane as compared to control. Also, the plant extracts promoted glucose uptake by the yeast cells. It was observed that the enhancement of glucose uptake was dependent on both the sample and glucose concentration. C. bonducella extract exhibited significantly higher (p≤0.05) activity than the extract of M. fragrans at all concentrations. The results of the study verified the hypoglycemic activity of the extracts of C. bonducella and M. fragrans. However, the observed effects exhibited by the extracts of seeds of C. bonducella and M. fragrans need to be confirmed by using different in vivo models and clinical trials for their effective utilization as therapeutic agents in better management of diabetes mellitus.

Hypoglycemic effects of Berberis aristata and Tamarindus indica extracts in vitro

ObjectiveThe objective of the present investigation was to evaluate the hypoglycemic potential of Berberis aristata and Tamarindus indica using various in vitro techniques. MethodsThe selected plant extracts were studied for their effects on glucose adsorption capacity, in vitro glucose diffusion, in vitro amylolysis kinetics and glucose transport across the yeast cells. ResultsIt was observed that both the plant extracts adsorbed glucose and the adsorption of glucose increased remarkably with an increase in glucose concentration. No significant (p⩽0.05) differences were observed between the adsorption capacities of B. aristata and T. indica. The results of amylolysis kinetic experimental model exhibited that the rate of glucose diffusion was increased with time from 30 to 180min and both the plant extracts demonstrated significant inhibitory effects on movement of glucose into external solution across dialysis membrane as compared to control. It was observed that the plant extracts also promoted glucose uptake by the yeast cells. Enhancement of glucose uptake was dependent on both the sample and glucose concentration. B. aristata extract exhibited significantly higher (p⩽0.05) activity than the extract of T. indica at all concentrations. ConclusionThe results of the study verified the hypoglycemic activity of the extracts of B. aristata and T. indica. However, the observed effects need to be confirmed using different in vivo models and clinical trials for their effective utilization as therapeutic agents.

In vitro hypoglycemic effects and starch digestibility characteristics of wheat based composite functional flour for diabetics.

The associations between chronic feeding of high level of soluble/insoluble fibers and low serum glucose levels have been well documented. In the present study, composite flours were formulated using psyllium, barley and oat at two different levels [WPOB-I = wheat flour (75%), psyllium (5%), oat (10%) and barley (10%), WPOB-II = wheat flour (60%), psyllium (10%), oat (15%) and barley (15%)]. Chapaties were prepared from all formulations and various starch fractions were analyzed using controlled enzymatic digestion. The digestibility characteristics were studied using amylolysis kinetics employing porcine pancreatic α-amylase in vitro. Results showed that both the variations (WPOB-I & WPOB-II) had acceptable sensory qualities and had significantly lower (p ≤ 0.05) values for total starch (TS), rapidly digestible starch (RDS), resistant starch (RS), starch digestibility index (SDI) and rapidly available glucose (RAG) compared to control. Between the two variations, WPOB-I showed better starch digestibility characteristics with significantly lower (p ≤ 0.05) starch digestibility index (SDI). In case of amylolysis kinetics, both the variations significantly (p ≤ 0.05) inhibited α-amylase as reflected by lower glucose diffusion and significantly higher (p ≤ 0.05) glucose dialysis retardation index (GDRI) compared to control. It is inferred that, consumption of the composite flours might be helpful in establishing stable blood glucose pattern due to the redistribution of nutritionally important starch fractions and inhibition of carbohydrate digestion in the gastrointestinal tract.

Health related functional characteristics and antioxidant potential of mucilage (dietary fiber) from Zizyphus mauritiana fruits

The composition of Zizyphus mauritiana mucilage (ZMM) and several properties related to its nutritional quality were investigated. The ZMM with good water holding capacity (25.21g/g), swelling capacity (19.34mL/g), glucose dialysis retardation index and in vitro amylolysis kinetics indicate that it may have the capability in controlling the diabetes. Structural characteristics of ZMM were analyzed using the Fourier transform infrared (FT-IR) spectroscopy. In addition, the ZMM has the strong antioxidant potency against ABTS (16,587.32mmol trolox equiv./g), DPPH (5.27g mucilage/g DPPH), hydroxyl (76.13%) and superoxide (85.12%) radicals due to the presence of polyphenols (25.54mg GAE/g mucilage). Besides, the ZMM also inhibited the acetylcholine esterase (86.89%), tyrosinase (47.01%), α-amylase (70.13%) and α-glucosidase (87.14%) enzymes. These properties make the crude mucilaginous fraction from Z. mauritiana fruit an excellent candidate of potential nutraceutical and functional food.

In vitro hypoglycemic effects of Albizzia lebbeck and Mucuna pruriens

ObjectiveTo verify the antidiabetic potential of stem bark of Albizzia lebbeck (A. lebbeck) and seeds of Mucuna pruriens (M. pruriens) using various In vitro techniques. MethodsThe plant extracts were studied for their effects on glucose adsorption, diffusion amylolysis kinetics and glucose transport across yeast cells. ResultsBoth the plant extracts adsorbed glucose and the adsorption of glucose increased remarkably with an increase in glucose concentration. No significant (P≤0.05) differences were observed between the adsorption capacities of A. lebbeck and M. pruriens. In amylolysis kinetic experimental model the rate of glucose diffusion was found to increase with time from 30 to 180 min, and both the plant extracts demonstrated significant inhibitory effects on movement of glucose into external solution across dialysis membrane as compared to control. The retardation of glucose diffusion by A. lebbeck extract was significantly higher (P≤0.05) than M. pruriens. These effects were reflected with higher glucose dialysis retardation index values for A. lebbeck than M. pruriens. The plant extracts also promoted glucose uptake by yeast cells. The rate of uptake of glucose into yeast cells was linear in all the 5 glucose concentrations used in the study. M. pruriens extract exhibited significantly higher (P≤0.05) activity than the extract of A. lebbeck at all concentrations. ConclusionsThe results verified the antidiabetic potential of A. lebbeck and M. pruriens. The hypoglycemic effect exhibited by the extracts is mediated by increasing glucose adsorption, decreasing glucose diffusion rate and at the cellular level by promoting glucose transport across the cell membrane as revealed by simple In vitro model of yeast cells.

Improved cold starch hydrolysis with urea addition and heat treatment at subgelatinization temperature

We explore how the presence of urea can influence the kinetics of amylolysis, with a long-term objective of developing practical and energy efficient bioconversion protocols. In this study, triticale and corn starches were hydrolyzed by a granular starch hydrolyzing enzyme with or without addition of urea and a pre-heating treatment at subgelatinization temperature. Differential scanning calorimetry showed that the gelatinization parameters of triticale and corn starches were negatively correlated with the urea concentration in the starch suspension. Addition of urea did not significantly affect starch amylolysis by the granular starch hydrolyzing enzyme at 30 °C. However when pre-heating at a higher yet sub-gelatinization temperature (50 °C for triticale and 61 °C for corn, 5 °C below the onset of starch gelatinization) for 30 min, the presence of urea greatly facilitated the amylolysis of both tricticale and corn starches. Scanning electron microcopy revealed starch granule mophological changes to a porous structure in residual starch granules/fragments rich in resistant starch. This means that the amylolysis pattern in the presence of urea was fundamentally changed, and urea disrupts starch hydrogen bonds effectively with heating treatment at a sub-gelatinization temperature. This treatment combination increased both starch hydrolysis rate and extent. Since extra energy was not necessary to gelatinize starch, this method may benefit starch and bio-enthanol industries to reduce the costs of starch hydrolysis.

In vitro hypoglycemic effects of Butea monosperma Lam. leaves and bark

The present study evaluated the antidiabetic potential of Butea monosperma Lam. Kuntze (Fabaceae) leaves and the stem bark using various in vitro techniques. The samples were studied for their effects on glucose adsorption, diffusion, amylolysis kinetics, enteric enzymes and glucose transport across yeast cells. Both the samples adsorbed glucose and the adsorption of glucose increased with increase in glucose concentration. The samples also inhibited movement of glucose across the dialysis membrane to varying degree in both glucose diffusion and amylolysis kinetic experiment models. B. monosperma leaves inhibited α-amylase, α-glucosidase and sucrase enzymes in succession to varying degrees, whereas the bark inhibited only α-amylase to a significant extent and slightly activated α-glucosidase and sucrase enzymes. The extracts of both leaves and bark promoted glucose uptake by yeast cells compared to control. Enhancement of glucose uptake was dependent on both the sample and glucose concentration. It was directly proportional to the sample concentration and inversely proportional to the molar concentration of the glucose. From the results of the study, it is inferred that, B. monosperma leaves and bark possesses antidiabetic activity. However, these effects need to be confirmed using in vivo models and clinical trials for its effective utilization as therapeutic agents.

The relation of physical properties of native starch granules to the kinetics of amylolysis catalysed by porcine pancreatic α-amylase

Various native starch granules were tested as substrates for porcine pancreatic α-amylase and enzyme kinetic parameters obtained from initial rate studies. Surface area per kg weight values and gelatinisation enthalpies (Δ H gel ) for the different granules were measured. Δ H gel was used as an indication of the degree of disorder within the starch granules. K m values, that are indicative of starch availability, decreased with the particle size but not in a linear fashion. The catalytic efficiency of amylase and the initial rate of catalysed hydrolysis were both significantly correlated with the surface area. Also, K m , catalytic efficiency ( k cat / K m ) and initial hydrolysis rate were all significantly correlated with Δ H gel . Thus the surface area of granules and the degree of order of starch have important influences on the initial rate at which native starch is digested by amylase. We believe that this is the first systematic report linking kinetic data with surface area and gelatinisation parameters of native starch.

In vitro studies on the hypoglycemic potential of Ficus racemosa stem bark

Medicinal plants have been reported to play an important role in modulating glycemic responses and have preventive and therapeutic implications. Several mechanisms have been proposed for the antidiabetic effect of medicinal plants such as inhibition of carbohydrate-metabolizing enzymes, manipulation of glucose transporters, beta-cell regeneration and enhancing insulin-releasing activity. The present investigation evaluated the possible mechanism of action through which Ficus racemosa stem bark (Moraceae) exerts its hypoglycemic effect using suitable in vitro techniques. Ficus racemosa bark (FRB) exhibited significantly higher (P < or = 0.01) glucose-binding capacity than wheat bran (WB) and acarbose (ACB) consequently showed significantly higher (P < or = 0.01) retardation of glucose diffusion compared to WB and ACB. In case of amylolysis kinetics the liberation of glucose was greatly inhibited by FRB, as reflected by a significantly lower (P < or = 0.01) glucose diffusion rate in the system containing FRB compared to the control and acarbose. Furthermore, FRB significantly increased (P < or = 0.01) the rate of glucose transport across the yeast cell membrane and also in isolated rat hemi-diaphragm. The findings indicate F. racemosa bark to possess strong hypoglycemic effect and hence can be utilized as an adjunct in the management of diabetes mellitus.

Enzymic susceptibility of starch from pasta

The aim of this work was to study the structural factors of pasta products that are responsible for their low glucose and insulin responses and incomplete intestinal absorption of starch in healthy subjects. Native starch extracted from durum wheat in the laboratory was used as reference. Starch granules in pasta of different sizes, cooked for different time periods, were completely gelatinized, as proved by differential scanning calorimetry and X-ray diffraction. Compared to native starch, susceptibility to α-amylase hydrolysis was always increased by cooking. Complete solubilization into oligosaccharides was obtained in 24 h for enzyme concentrations higher than 600 nkat/ml with 17 mg starch per ml. Smaller pasta size and increased cooking time led to higher susceptibility. When pasta structure was destroyed by grinding, starch was solubilized completely in less than 30 min by α-amylase hydrolysis. Limited swelling of starch granules during cooking and encapsulation by the protein network were partly responsible for the slow amylolysis kinetics. An estimation of the susceptibility towards α-amylase of gelatinized starchy products is proposed, based on the minimal concentration of α-amylase necessary to obtain the solubilization of the easily degradable fraction.

The Kinetics of Amylolysis. Part II. Determination of Reactive and Non‐Reactive Enzyme Substrate Encounters in beta‐Amylolysis

AbstractThe ratio of the reactive and non‐reactive enzyme substrate encounters was determined from the data of measurements obtained at the β‐amylolysis of amylose of DP = 250 labelled with 14C at its non‐reducing chain end, taking into account the self‐inhibiting effect of the inner segments of the amylose chain. During the β‐amylolysis the number of bond splittings was 14.6 per effective enzyme substrate encounters, and only 0.405 when also the inactive encounters are taken into account, under the chosen experimental conditions.From these values and from the kinetic constants, the actual rate constants of the formation and dissociation of both reactive and inactive enzyme substrate complexes were determined.

Über die Kinetik der Amylolyse. Teil V. Untersuchung der SH‐Gruppen der Weizen‐beta‐Amylase und Nachweis der induzierten Anpassung

AbstractIn presence of substrate the inhibiting effect of thiol reagents is vastly decreasing. The enzyme molecule undergoes certain structural changes as a cause of „induced adaptation”︁, thus rendering the internal SH‐groups inaccessible. Those free SH‐groups on the surface play no significant role in the reaction catalyzed by the enzyme.On the Kinetics of Amylolysis. Part V. Examination of the SH‐Groups of Wheat‐β‐Amylase — Proof of Induced Adaptation.During titration of wheat‐β‐amylase with p‐chlorine mercury benzoate (pCMB) it was stated that the enzyme protein contains four sulfhydryl‐groups (SH‐groups) per molecule two of which are lying freely on the surface, whereas two are „hidden”︁. In absence of substrate thiol reagents instantly react with the SH‐groups. pCMB and N‐ethyl maleinimide cause an almost complete inhibition which is non‐competitive in the case of pCMB as was demonstrated in reaction kinetic tests.

The kinetics of amylolysis : Part III. Effect of self-inhibition on the kinetic constants of beta-amylolysis

For beta-amylolysis, the number of bonds split per effective enzyme-substrate encounter has been determined with amyloses having various degrees of polymerization (d.p.), labelled with 14C. The degradation is shifted in the direction of the “single chain mechanism” with increasing d.p. By taking into account the “inactive, non-reacting” collisions leading to “self-inhibition”, the number of bonds split per “reactive” and “non-reactive” enzyme-substrate encounter was established. From these data, and from the inhibition constant of the inner D-glucose residues of the amylose chain, in conjunction with the Michaelis constant extrapolated to d.p.  4, the actual rate constants for the formation and dissociation of the “reactive” enzyme-substrate complexes and for the formation and dissociation of the “non-reactive” enzyme-substrate complexes were determined. It was found that the rate constants for the formation of the “reactive” complexes decrease slowly with the increase of “self-inhibition”, whereas those for the dissociation of these complexes decrease to a greater extent.