Mode of Inhibition of α-Glucosidase and α-Amylase by ... · PDF fileMaqui...
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Mode of Inhibition of α-Glucosidase and α-Amylase by Polyphenol-Enriched Extracts of Maqui (Aristotelia chilensis) Francisca Acevedo a , Mónica Rubilar a,b* , Barbara Palma a , Carolina Shene a,b a Center of Food Biotechnology and Bioseparations, BIOREN, Universidad de La Frontera, Casilla 54-D, Temuco, Chile ([email protected]) b Technology and Processes Unit, CGNA, Universidad de La Frontera, Casilla 54-D, Temuco, Chile ([email protected]) INTRODUCTION Polyphenolic compounds are known to be useful in formulating of nutritional or medicinal supplements for the treatment of several diseases. An important activity of polyphenols is the inhibition of digestive enzymes, especially carbohydrate-hydrolyzing enzymes such as α-amylase and α-glucosidase. Inhibitors of these enzymes are able to retard carbohydrate digestion, thus causing a reduction in glucose absorption rate. Effective α-amylase and α-glucosidase polyphenol- type inhibitors from natural resources have been reported to be useful in reducing postprandial hyperglycemia [1]. To our knowledge, the inhibition mode of maqui leaf crude extract against α- glucosidase and α-amylase has not been reported yet. MATERIALS & METHODS Leaves of maqui was collected from the Andes mountains in the Araucanía Region (Chile). The collected material was dried at 35 °C, ground and sieved. The sample (5 g) was macerated with ethanol (50% v/v in water, solvent-to-solid ratio of 5:1) at room temperature and filtered. Filtrate was concentrated in a rotary evaporator at 35°C and lyophilized. The dry powder was dissolved in ethanol 50% v/v. In order to examine the inhibition mode of maqui leaf crude extract, α–amylase and α-glucosidase activities were measured with increasing concentrations of substrate in the absence and presence of maqui leaf extract at different concentrations. Alpha-amylase activity was quantified by measuring the maltose equivalents released from starch at 540 nm. Alpha-glucosidase activity was quantified by measuring the para-nitrophenol equivalents released from pNPG at 400 nm. The Michaelis-Menten constant (K m ), maximum enzyme reaction rate (V max ) and the inhibition mode of maqui leaf crude extract on the α-amylase-catalyzed hydrolysis of starch and on the α- glucosidase-catalyzed reaction of pNPG were estimated using Lineweaver–Burk plots. In this study, the initial velocity ‘v’ of the hydrolysis reactions catalyzed by α- amylase was measured at various substrate concentrations [S] (0.2 – 1% starch) in the absence and presence of several maqui leaf crude extract concentrations [I] (10 – 100 ppm).
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Mode of Inhibition of α-Glucosidase and α-Amylase by Polyphenol-Enriched Extracts of
Maqui (Aristotelia chilensis)
Francisca Acevedoa, Mónica Rubilara,b*, Barbara Palmaa, Carolina Shenea,b
aCenter of Food Biotechnology and Bioseparations, BIOREN, Universidad de La Frontera, Casilla 54-D, Temuco, Chile ([email protected])
bTechnology and Processes Unit, CGNA, Universidad de La Frontera, Casilla 54-D, Temuco, Chile ([email protected])
INTRODUCTION
Polyphenolic compounds are known to be useful in formulating of nutritional or medicinal supplements for the treatment of several diseases. An important activity of polyphenols is the inhibition of digestive enzymes, especially carbohydrate-hydrolyzing enzymes such as α-amylase and α-glucosidase. Inhibitors of these enzymes are able to retard carbohydrate digestion, thus causing a reduction in glucose absorption rate. Effective α-amylase and α-glucosidase polyphenol-type inhibitors from natural resources have been reported to be useful in reducing postprandial hyperglycemia [1]. To our knowledge, the inhibition mode of maqui leaf crude extract against α-glucosidase and α-amylase has not been reported yet. MATERIALS & METHODS
Leaves of maqui was collected from the Andes mountains in the Araucanía Region (Chile). The collected material was dried at 35 °C, ground and sieved. The sample (5 g) was macerated with ethanol (50% v/v in water, solvent-to-solid ratio of 5:1) at room temperature and filtered. Filtrate was concentrated in a rotary evaporator at 35°C and lyophilized. The dry powder was dissolved in ethanol 50% v/v. In order to examine the inhibition mode of maqui leaf crude extract, α–amylase and α-glucosidase activities were measured with increasing concentrations of substrate in the absence and presence of maqui leaf extract at different concentrations. Alpha-amylase activity was quantified by measuring the maltose equivalents released from starch at 540 nm. Alpha-glucosidase activity was quantified by measuring the para-nitrophenol equivalents released from pNPG at 400 nm. The Michaelis-Menten constant (Km), maximum enzyme reaction rate (Vmax) and the inhibition mode of maqui leaf crude extract on the α-amylase-catalyzed hydrolysis of starch and on the α-glucosidase-catalyzed reaction of pNPG were estimated using Lineweaver–Burk plots. In this study, the initial velocity ‘v’ of the hydrolysis reactions catalyzed by α- amylase was measured at various substrate concentrations [S] (0.2 – 1% starch) in the absence and presence of several maqui leaf crude extract concentrations [I] (10 – 100 ppm).
RESULTS AND DISCUSSION
Km value for α-amylase was found to be 1.8% starch with a Vmax value of 6.8 µM min-1. The results indicate that maqui leaf crude extracts act as mixed-type inhibitors, binding to either α-amylase (E) or enzyme-substrate (ES) complex, resulting in a decrease in the apparent affinity of α-amylase for starch (increase Km) and a decrease in the apparent Vmax. A similar type of Acarbose inhibition and for the two Acarbose analogues for porcine pancreatic α-amylases was reported by Yoon and Robyt [5]. Similar behavior appears reported in literature for finger millet seed coat phenolics, which behave as non-competitive inhibitors on pancreatic α-amylase, being Km value for this enzyme about 1% starch [3]. The v of the hydrolysis reactions catalyzed by α-glucosidase was measured at various substrate concentrations [S] (2-5 – 15-0 mM pNPG) in the absence and presence of several maqui leaf crude extract concentrations [I] (0.05 – 5-0 ppm). The Km for α-glucosidase was found to be 3.5 mM starch with a Vmax value of 0.25 mM min-1. The results of this study indicate that binding of phenolic compounds to enzyme affected the velocity of α-glucosidase reaction rate, proportionally to the concentration of the phenolic compounds in the reaction mixture, not modifying Km value. Thus, a non-competitive inhibition by maqui leaf crude extracts upon α-glucosidase-catalyzed pNPG hydrolysis was found in this study. Reversible non-competitive inhibition of α-glucosidase is reported in literature for aqueous extracts from the gall of Rhus chinensis [2]. Alpha-glucosidase inhibition by several flavonoids has been reported as mixed-type and almost non-competitive [4]. CONCLUSION
Furthermore, the evaluation of digestive enzyme inhibition in the extract represents a preliminary approach to the potential biological properties of maqui leaves, which are used in traditional medicine. REFERENCES
[1] Gao H., Huang Y.N., Gao B., Xu P.Y, Inagaki C.; Kawabata J. 2008. α-glucosidase inhibitory effect by the flower buds of Tussilago farfara L. Food Chemistry, 106, 1195-1201.
[2] Shim Y.-J., Doo H.-K., Ahn S.-Y., Kim Y.-S., Seong J.-K., Park I.-S., Min B.-H. 2003 Inhibitory effect of aqueous extract from the gall of Rhus chinensis on alpha-glucosidase activity and postprandial blood glucose. Journal of Ethnopharmacology, 85, 283-287.
[3] Shobana, S.; Sreerama, Y.N., Malleshi, N.G. 2009. Composition and enzyme inhibitory properties of finger millet (Eleusine coracana L.) seed coat phenolics: mode of inhibition of a-glucosidase and pancreatic amylase. Food Chemistry 115, 1268–1273.
[4] Tadera K.; Minami Y.; Takamatsu K.; Matsuoka T. 2006.Inhibition of α-glucosidase and α-amylase by flavonoids. Journal of Nutritional Science and Vitaminology. 52, 149-153.
[5] Yoon S.-H.; Robyt J.F. 2003Study of the inhibition of four alpha amylases by acarbose and its 4IV-a-maltohexaosyl and 4IV-a-maltododecaosyl analogues. Carbohydrate Research, 338, 1969-1980.
