Abstract

Diabetes is a severe chronic disease caused by insufficient production of or resistance to insulin, which results in abnormal metabolism of carbohydrates, fats, and proteins. Obesity has been identified as an important variable in diabetes. The inhibition of -amylase activity has been suggested as a strategy for diabetes and obesity management by reducing sugar levels in blood. In this study we examined the inhibition of -amylase by methanolic and aqueous extracts of Tapeinochilus ananassae, the flavonoid Quercetin, and Acarbose. T. ananassae has ethnomedicinal use in Puerto Rico and is known as insulina. In this study, we determined the percent of reaction of -amylase following the production of maltose from starch at = 540 nm after reaction with DNS using a colorimetric assay. The percent inhibition and IC50 of extracts and positive controls was calculated from the followings concentrations: Quercetin [22.5 360 g/mL], Acarbose [5-80 g/mL], and T. ananassae [20.45 - 327.14 g/mL] showed a direct response with concentration. IC50 values for Quercetin, Acarbose, and T. ananassae were 396.8 g/mL, 90.8 g/mL, and 365.8 g/mL respectively. We found that aqueous extracts of T. ananassae showed an inverse concentration-inhibition response. These results suggest that the extracts could be promoting the (starch + -amylase = simple sugars) reaction that has been reported also Lypinus luteus and Pharbitis nil extracts. Therefore, because the herbal remedy is a tea, -amylase inhibition with T. ananassae is not a therapeutic approach.

Future Work
Study -amylase inhibition of methanolic and aqueous extracts from Rhoeo spathacea, Syszygium jambos and Costus sp. , plants with ethnomedicinal use in Puerto Rico.

References
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Methods
Plant Material Plant leaves were lyophilized and extracted with methanol for 24 h using a soxhlet apparatus. The extract was evaporated to dryness with a rotary evaporator and tested without further purification.(4) In aqueous extract we used the decoction technique. Maltose Calibration Curve We prepared maltose standard solutions 0 - 0.05% w/V [0.01, 0.02, 0.03, 0.04, 0.05 w/V] combining 5.0, 10.0, 15.0, 20.0, 25.0 L of maltose solution (1.8% w/V) and 895.0, 890.0, 885.0, 880.0, 875.0 L of distilled water respectively, and 100.0 L of DNS color reagent. The mixture was placed in an 85C water bath during 15 minutes. The [maltose] determined measuring the absorbance at 540nm. The linear regression of the calibration curve was used to determine [maltose] in the presence of inhibitors and plant extracts.(4-6) Assays were performed in triplicate. -AmylaseInhibitionBioassay -Amylase inhibition bioassay was performed at five different concentration s(327.14, 163.57, 81.79, 40.89, 20.45g/mL) of T. ananassae methanolic and aqueous extract and DMSO as control. (1-2, 4-5, 7-12) In the control experiment 100 L of -amylase solution (0.5% w/V), and 100L of DMSO was incubated for 5 minutes at 37C, and then mixed with 400L of starch solution, and 100L of distilled water. The resulting solution was incubated for 3 minutes at r.t. and place into an 85C water bath during 15 minutes after the addition of 100L of DNS color reagent. Before we add 900 L of distilled water. The bioassay was performed in triplicate. After 15 minutes, the absorbance at 540nm was determined. The same procedure was followed for Acarbose [80.0 5.0g/mL], and Quercetine [360.0 22.5g/mL]. -Amylase activity (the production of maltose) was determined by measuring the absorbance of the solution at 540nm. From the absorbance, the % of maltose generated was calculated from the equation obtained from the maltose calibration curve. Percent of residual reaction was calculated as (maltose in test/ maltose in control) x 100.

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Introduction and Specific Aim

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Diabetes mellitus (DM) is a chronic disease caused by acquired deficiency in insulin secretion or by decreased responsiveness of the organs to secreted insulin. This deficiency results in hyperglycemia. DM affects about 5% of the global population and is one of the most costly and burdensome chronic diseases.1 There are two forms of diabetes: Type 1 diabetes includes cases which can be attributed to an autoimmune process and Type 2 includes the common major form of diabetes which results from defects in insulin secretion. The only therapy for Type 1 is the substitution of insulin. The treatments for Type 2 are many and diverse, and include strategies as the inhibition of -amylase.2 -Amylase inhibitors are also known as starch blockers. Starches are complex carbohydrates that cannot be absorbed unless they are first broken down by the digestive enzyme amylase and other secondary enzymes.3 The inhibitors of these enzymes delay carbohydrate digestion and prolong overall carbohydrate digestion time, causing a reduction in the rate of simple sugars absorption. Two of these inhibitors are: Acarbose and Quercetin. Acarbose is an anti-diabetic drug and Quercetin a flavonoid found in plants. Many plants have been investigated for their potential to reduce the production of simple sugars from carbohydrates.1 They play an important role in the treatment and management of diabetes, particularly in developing countries where most people have limited access to primary health care.4 In this study, we have selected aqueous and methanolic extracts of T. ananassae, known locally as insulina, to evaluate its capacity to inhibit -amylase.

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Results and Discussion

Acarbose is a known -amylase noncompetitive inhibitor. This class of inhibitors reduces the efficiency of the enzyme binding to the enzyme in a site different to its active site. This binding reduces the velocity of the enzymatic reaction. The positive controls used in this experiment were Quercetin and Acarbose. The residual activity exhibited by the positive controls, was inversely proportional to their concentration. That is, when the concentration of the inhibitor increases the concentration of maltose decreases. Therefore, inhibition of -Amylase activity was observed (Figures 1 and 2). Quercetin inhibition was significant at the concentrations tested, (IC 396.8g/mL). Acarbose inhibition in this study was less than reported previously 4 (IC 90.8g/mL vs. IC 50 50 50 80.0g/mL), due to the presence of impurity and uncertainty of the experiment. (Mp : 90 dec)
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Inhibition of -Amylase activity of T. ananassae methanolic extract produced an IC 365.8g/mL (Figure 3). The activity observed for the extract suggests that -amylase inhibition might be the mechanism that control simple sugars levels in the blood when the plant used as a herbal medicine. An increase in -amylase activity (more maltose is produce in the presence of the extract than in the control) was observed when T. ananassae aqueous extract was tested (Figure 4). The increase observed in the aqueous extract suggests the presence of active ingredients that enhance the enzyme activity or that reducing sugars are present in the extract. The results obtained for the inhibition of -amylase suggests that secondary metabolites of different polarities are present in the extract. The difference in activities in the extracts might result from the polarity of the extraction solvent.
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Plant and Inhibitors Under Study

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Acknowledgements

Figure 1. Residual Activity Percent to Determine the Acarbose IC50

Figure 3. Residual Activity Percent to Determine T. ananassae Methanolic Extract IC50

T. ananassae

Acarbose

Quercetin

Program Goal
To develop in vitro bioassay panels for diabetes, asthma and oxidative stress to assess the efficacy, safety and bioavailability of plant extracts and botanical supplements as herbal therapies.

Figure 2. Residual Activity Percent to Determine the Quercetin IC50

Figure 4. Residual Activity Percent of T. ananassae Aqueous Extract