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Inhibitory effect of dates-extract on α -Amylase and α-glucosidase
enzymes relevant to non-insulin dependent diabetes mellitus
Sulaiman Al-Zuhair*, Ali Dowa
idar and Hassan Kamal
Received: 4 April 2010 / Received in revised form: 9 June 2010, Accepted: 9 June 2010, Published online: 7 July 2010 Abstract
α-Amylase and α-glucosidase are key enzymes involved in available anti-diabetic drugs used to treat non-insulin dependent carbohydrates breakdown and intestinal absorption, respectively. diabetes mellitus NIDDM, such as Acarbose, strongly inhibits both Inhibition of these enzymes hinders blood glucose level increase enzymes. However, patients using Acarbose usually suffer from after a carbohydrate diet and can be an important strategy in the abdominal distention, flatulence, meteorism and possibly diarrhea management of non-insulin-dependent diabetes mellitus (NIDDM). (Bischoff, 1994). These side effects are caused by the excessive A main drawback of currently used inhibitors is their side effects inhibition of pancreatic α-amylase resulting in the abnormal such as abdominal distention, flatulence, meteorism and diarrhea, bacterial fermentation of undigested  carbohydrates in the colon caused by excessive inhibition of pancreatic α-amylase resulting in (Bischoff, 1994; Horii, 1987). Therefore it is attractive to find a abnormal bacterial fermentation of undigested carbohydrates in the substance that has a strong inhibitory activity against α-glucosidase, colon. Natural inhibitors from plants have shown lower inhibitory but minor effect on α-amylase activity (Kwon et al., 2006). As dates effect against α-amylase activity and a stronger inhibitory activity represent large portion of sugar source in Middle Eastern diet, it against α-glucosidase and can be used as effective therapy for would be advantages to find out if they have positive effect on NIDDM with minimal side effects. In this work dates-extract (DE) NIDDM. The results of this work will open the door for further inhibitory effect on α-amylase and α-glucosidase has been assessed. investigation into the active agents that cause the inhibitory effect, The inhibition percentages on α-amylase and α-glucosidase were in which can be extracted and used as a natural drug. the range of 6-24% and 54%, respectively. The results clearly show the NIDDM treatment potential of DE. Chemicals and Methods
Keywords: Diabetes, dates-extract, α-amylase, α-glucosidase,
Chemicals
Dates-syrup was purchased from local market and dates-pits Introduction
granules were obtained from Al-Saad Dates Processing Factory, UAE. α-Amylase from Aspergillus oryzae, 1.5 units mg-1 and α- -Amylase and α-glucosidase are enzymes involved in starch glucosidase from Saccharomyces cerevisiae Type I, lyophilized breakdown and intestinal absorption, respectively. The first enzyme powder with ≥10 units mg-1 were purchased from Sigma-Aldrich, is involved in the digestion of carbohydrates to produce simpler USA. All other chemicals were of analytical grade and purchased saccharides, whereas the second is involved in their absorption. It is believed that inhibition of the two enzymes would result in a lower blood glucose levels after a rich carbohydrate diet. The current Dates- and dates-pits extracts preparation
Dates-extract (DE) was prepared by mixing 50 ml of dates-syrup Sulaiman Al-Zuhair*, Ali Dowaidar and Hassan Kamal
thoroughly with to 50 ml of distilled water. The mixture was then centrifuged at 4000 rpm (ICE CL31 Multispeed Centrifuge, Department of Chemical and Petroleum Engineering, UAE Thermoscientific, USA), and the supernatant was vacuum-filtered University, Al-Ain, United Arab Emirates (Shel Lab, USA). Vacuum was generated using jet pump and the filter paper used was Whatman-Gade 1(11 μm). *Tel: +9713713363; Fax: +97137624262 Email: [email protected] Dates-pits extract (DPE) was prepared by initially washing the granules. Then they were dried and course grinded using Sanyo, Japan grinder and then fine grinded using Moulinex, France grinder. The particles were then screened in Pascal Eng. Co. Ltd., England using mesh 70-120, which gives particle sizes in the range of 125- 212 μmand screened. 20 g of the collected granules were dispersed in 50 ml distilled water in a screw capped bottle. The bottle was increased at higher substrate concentration reaching 24% at initial kept on a shaker (WSB-30, Korea) at 350 rpm for 24 hours to reach equilibrium. The mixture was then vacuum-filtered (Shel Lab, USA). -Amylase assay
The reactions took place in ten test tubes, each containing 1 ml distilled water (or DE solution), 1 ml of 100 g l-1 starch solution in 2 mM phosphate buffer solution (pH 6.9) and 1 ml of 0.05 g l-1 α- amylase in phosphate buffer solution. The test tubes were incubated at 37 oC (which represent normal body temperature). The enzymatic reaction was stopped by adding 50 μl of 1 M HCl to each test tube at different times in the range of 10 to 100 seconds, with 10 minutes intervals between the test tubes. The reducing sugar formed in the reaction was measured by 3,5-dinitrosalicylic acid (DNS) method using maltose as the standard (Miller, 1959) and was used to determine the activity of α-amylase. One unit of α-amylase was defined as the amount of enzyme required to produce 1 µmol of reducing equivalents per minute from soluble starch under the assay conditions. The experiment was then repeated at different initial amounts of substrate in the range of 33 g l-1 to 66 g l-1. α-Glucosidase assay
Time, t (min)
A modified method to the one used by (Kwon et al., 2008) has been Figure 1: Effect of DE on enzymatic production of deviation maltose at starch concentration of 33 g l-1 (o without DE and with DE) with time. α-Glucosidase activity was assayed using 1 ml of 0.35 mM p-nitrophenyl-α-D-glucopyranoside solution in 100 μmol of α-Glucosidase inhibition
acetate buffer (pH 4.5) mixed with 1 ml of distilled water (or DE or DPE solutions), 0.5 ml of 200 mM Na The rate of p-phenol production was determined using different α-glucosidase solution. The reaction took place in a special vial initial p-nitrophenyl-α-D-glucopyranoside solution, with the placed in the spectrophotometer, and the absorbance at 405 nm was presence of DE and DPE and in absence of any inhibition. For recorded as a function of time. The absorbance was compared to reference, the experiment was also repeated in absence of α- that of standard solutions of p-nitrophenol and the results were used glucosidase. The results at initial substrate concentration of 0.35 to determine the activity of α- glucosidase. One unit of α- mM are shown in Fig. 2. All experiments were done in duplicate, glucosidase was defined as the amount of enzyme required to and the reproducibility of the results is confirmed from the small error bars shown in the figure. The results show that the presence of p-nitrophenol per minute under the assay conditions. The experiment was then repeated at different initial DE has significant effect on the activity of α-glucosidase. On the amounts of substrate in the range of 0.07 mM to 0.42 mM. The other hand, only slight inhibition effect was observed in the experiment was also run at substrate concentration of 0.35 mM in presence of DPE. Table 2 shows α-glucosidase inhibition of DE and absence of α-glucosidase as a reference. DPE. It can be seen that the presence of DE significantly inhibited α-glucosidase, and reduced its activity by 54%, which is almost Results and Discussion
equal to the absence of enzyme. This indicated that the DE almost completely inhibited the enzyme. On the other hand, date-pits α-Amylase inhibition
extract, inhibited the enzyme much less and percentage reduction of The rate of reduced sugar production was determined using different only 30% was observed. Similar results were observed at other initial starch concentrations, with and without the presence of DE. initial starch concentration in the range of 0.07 - 0.42 mM, and the To eliminate the effect of reduced sugar initially found in the date- extract, the results were presented in terms of deviation maltose Table 1: Substrate concentration effect on α-amylase activity
concentration, P, defined as difference between the concentration of
reduced sugar at anytime and that at time zero. An example of the -amylase activity(U)
percentage
[S] (g l-1)
deviation maltose production with time, with and in absence of DE, reduction
Without DE
at initial starch concentration of 33 g l-1 is shown in Fig. 1. All experiments were done in duplicate, and the reproducibility of the results is confirmed from the small error bars shown in the figure. Although slight inhibition effect was observed, the results show that the presence of DE has insignificant effect on the activity of α-amylase. Similar results were also observed at other initial starch The use of DE in the treatment of NIDDM concentration in the range of 33 to 66 g l-1. Results indicated that DPE slightly inhibits the activity of α- α-Amylase activity was determined at different initial substrate amylase. The inhibition percentage was in the range of 6-24%, concentrations and the results are shown in Table 1, with and depending on the initial substrate concentration. The inhibition without the presence of DE. It can be seen that the presence of DE effect of DE was less than that of egg-plant extract used by Kwon et slightly inhibited α-amylase with less than 10% inhibition at al. (2008), which was in the range of 14-38%. This is a favourable concentrations 33 and 50 g l-1. However, the inhibition effect Conclusion

The inhibition effect of DE on the activity of α-amylase and α-
glucosidase has been experimentally assessed. The results show that DE inhibits α-glucosidase more than it does on α-amylase, which is a positive result. The findings of this work clearly show the DE Acknowledgment
The authors would like to acknowledge the financial support provided by the Research Affairs at the UAE University. p
References

Bischoff H (1994) Pharmacology of glucosidase inhibitor. Eur J Time, t (min)
Horii S (1987) Synthesis and α-D-glucosidase inhibitory activity of Nsubstituted valiolamine derivatives as potent oral antidiabetic Figure 2: DE and DPE effect on p-phenol production, at initial substrate
concentration of 0.35 mM (o without enzyme, with DE, with DPE and Kwon YI, Apostolidis E, Shetty K (2008) In vitro studies of eggplant (Solanum melongena) phenolics as inhibitors of key enzymes relevant for type 2 diabetes and hypertension. Biores result, since as explained earlier, the excessive inhibition of α- amylase results in abnormal bacterial fermentation of undigested Kwon YI, Vattem DV, Shetty K (2006) Evaluation of clonal herbs carbohydrates in the colon, which intern results in abdominal of Lamiaceae species for management of diabetes and distention, flatulence, meteorism and possibly diarrhea (Bischoff, hypertension. Asia Pac J Clin Nutr 15:107–118 1994). On the other hand, it was found that DPE strongly inhibits Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428 Rosetti LL, Giacarri A, Defronzo RA (1990) Glucose toxicity. Shetty K, Curtis OF, Levin RE, Wikowsky R, Ang W (1995) Prevention of verification associated with in vitro shoot culture of oregano (Origanum vulgare) by Pseudomonas spp. J Plant Initial Substrate Concentration, [S]o (mM)
Figure 3: Substrate concentration effect on α-glucosidase activity
( without inhibitors, with DPE, with DE)
the activity of α- glucosidase and renders it almost inactive. The inhibition percentage using DE is found to be 54%, which is higher than the egg plant extract inhibition which was in the range of 5-46% (Kwon et al., 2008). Table 2: Effect of inhibitors on α-glucosidase activity
Percentage
α-glucosidase activity(U)
reduction

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