Inhibition Effects of Blueberries on α-Glucosidase

By Joshua Bernadin

 

Abstract

Type-2 diabetes is an epidemic. Blueberries could hold the key to a new natural form of prevention and possible treatment. The antioxidants in blueberries could inhibit the enzyme α-glucosidase, which can lower blood sugar spikes. An in vitro assay was performed to study the inhibition effects of blueberries on α-glucosidase. The IC₅₀ was found to be 1.0 mg/mL.

Introduction

Type-2 diabetes is an epidemic level disease. Of the 38 million Americans who have diabetes, 90% of them have type-2 (1, 2). Type-2 diabetes is characterized by a resistance to a hormone called insulin which aids in the transfer of sugar from the blood into cells for usage and storage. A resistance to insulin causes blood sugar spikes that can lead to loss of eyesight, nerve damage, and many other problems. Better lifestyle choices like diet and exercise have been proven to help prevent type-2 diabetes. However, due to genetic predisposition to contracting type-2 diabetes and the number of people who have it already, further prevention measures and possible treatments are needed.

There are many treatments available in today’s market for type-2 diabetes (1, 2). They all have their benefits and disadvantages, but they are all effective. Insulin injections introduce more insulin into the body to go past the resistance in type-2 diabetics. These injections are taken daily and are painful due to the injection happening over the pancreas. Metformin is a drug that lowers the glucose production in the liver. If not taken with food, it can cause GI issues. DPP-4 inhibitors stop the breakdown of GLP-1 and GIP hormones. These hormones regulate glucose levels in the body. There are different agonists for GLP-1 and GIP receptors, but these cause weight loss. People started using these receptor agonists as weight loss supplements leading to a shortage. Although all of these treatments are effective, most of them have side effects and are very expensive. In 2022, over $400 billion was spent on these treatments creating a need for a cheaper alternative.

α-Glucosidase inhibition was used for type-2 diabetes treatment. α-Glucosidase is an enzyme that resides in the small intestine that breaks down complex carbohydrates into glucose through hydrolysis (3). In type-2 diabetics, this leads to sharper blood sugar spikes due to the absorption of glucose. Drugs, like acarbose with an IC₅₀ of 2 µg/mL, completely inhibits the activity of α-glucosidase. This has the consequence of making these complex carbohydrates act as fiber, leading to GI issues. In order to use α-glucosidase inhibition for treatment and/or management of type-2 diabetes, partial inhibition of α-glucosidase is key.

Blueberries may hold the key to this partial inhibition. Blueberries are rich in polyphenolic antioxidants like flavonoids and anthocyanins. Previous experimentation has shown that these antioxidants have neuronal protective properties that could aid in the prevention of Alzheimer’s disease (4-7). This antioxidant activity could be used on α-glucosidase as preventative care or a possible treatment for type-2 diabetes.

Experimental

Blueberry Extract

The antioxidants in blueberries were extracted using a solvent mix of 40:40:19:1 of acetone, methanol, water, and formic acid. 500 g of fresh blueberries yielded 778 mg of the extract. This extraction created a dark violet powder (6, 7).

α-Glucosidase Inhibition Assay

The inhibition of α-glucosidase was determined in a 6.8 pH phosphate buffer in the presence and absence of the blueberry extract at 1, 0.5, 0.25, and 0.125 mg/mL concentrations in a final volume of 100 µL. Acarbose was used as a positive control. α-Glucosidase was incubated with the blueberry extract for 30 minutes in a dark area. Para-nitrophenol-α-D-glucoside (pNPG) was added to measure the activity of α-glucosidase. These samples were placed in a 96 well microplate. As soon as pNPG was added, the samples were placed in a spectrophotometer at 410 nm measuring the kinetic activity for an hour using SpectraMax M5 plate reader (Molecular Device, Sunnyvale, CA). Measurements were taken every minute. Using the average rates of absorption, the inhibition % was calculated using the following equation:

The inhibition assay was used to calculate the IC₅₀ of the blueberry extract. All measurements had multiple runs with n= 5-8.

Results

The α-glucosidase activity assay is based on the hydrolysis of pNPG, which release the glucose and p-Nitrophenol (yellow color) with absorbance at 410 nm. As shown in Figure 1, α-glucosidase showed strong activity of hydrolysis of pNPG, and acarbose, a known inhibitor of α-glucosidase completely inhibits the activity of α-glucosidase. The assay was used to examine the inhibition effect of the blueberry extract on α-glucosidase.

Figure 1: Kinetics of α-glucosidase activity assay using pNPG as the substrate with monitoring absorbance at 410 nm.

Figure 2: These figures shows the experimental samples before (left) and after (right) analysis in the spectrophometer. From top to bottom, the rows are enzyme control and enzyme with respective contractions of blueberry extract.

As shown in Figure 2, in samples containing the blueberry extract, little color change was reported. Through the rate of absorption given by the spectrophotometer, inhibition of α-glucosidase by blueberry extract was shown in a concentration dependent manner (Table 1 and Figure 3).

Table 1: Percentage inhibition of α-glucosidase by different concentration of blueberry extract

Figure 3: This graph shows how the inhibition % changes as concentration changes.

The IC₅₀ of blueberry extract was calculated through the concentration dependent inhibition curve (Figure 3), and was estimated to be 1 mg/mL of the extract, which is equivalent to 0.64 g/mL of fresh blueberries.

Conclusion

The inhibition of α-glucosidase was seen at each concentration tested. The IC₅₀ was measured to be 1 mg/mL of the blueberry extract, equivalent to 0.64 g/mL of fresh blueberries. This shows that the blueberry extract, although not as potent as acarbose, can effectively inhibit α-glucosidase partially. This partial inhibition can be the key to future preventative care and treatment for type-2 diabetes.

If given the chance, future studies would measure inhibition kinetics and mechanisms to test the fidelity of the blueberry extract and which antioxidants in the extract cause the α-glucosidase inhibition. This would lead to in vivo studies to test if blood sugar spikes are lessened with the active antioxidant. Afterwards, the blueberry extract’s inhibition effects would be tested on other enzymes and oxidative species that cause other issues. In conclusion, type-2 diabetes may find a new preventative measure or treatment through blueberries. For now, a few blueberries after a carb heavy meal may go a long way for preventing type-2 diabetes.

 

References

American Diabetes Association. “What Are My Options for Type 2 Diabetes Medications? ADA.” Diabetes.org, American Diabetes Association, diabetes.org/health-wellness/medication/oral-other-injectable-diabetes-medications.

Centers for Disease Control and Prevention. “Type 2 Diabetes.” Centers for Disease Control and Prevention, 18 Apr. 2023, www.cdc.gov/diabetes/basics/type2.html.

Daou, Mariane, et al. “In Vitro α-Glucosidase Inhibitory Activity of Tamarix Nilotica Shoot Extracts and Fractions.” PLOS ONE, vol. 17, no. 3, 14 Mar. 2022, p. e0264969, https://doi.org/10.1371/journal.pone.0264969.

Costa, Sophia (2022). “Neuronal Protective Effects of Blueberries against Oxidative Stress on Human Neuroblastoma Cells and Anti-Amyloidogenic Properties.” Thesis, Chemistry and Biochemistry, University of Massachusetts Dartmouth. umassd.primo.exlibrisgroup.com/discovery/delivery/01MA_DM_INST: umassd_library/12133216540001301.

Roderick, Chelsea (2022), Phytochemical profiling of blueberries and their neuronal protection through the inhibition of tyrosinase and acetylcholinesterase: a thesis in Chemistry and Biochemistry, University of Massachusetts Dartmouth.

Samani, Pari (2022). “Anti-inflammatory Properties and Neuroprotective Effects of Blueberries – an implication for the prevention of Alzheimer’s disease.” Dissertation in Chemistry and Biochemistry, University of Massachusetts Dartmouth.

Samani, P.; S. Costa; S. Cai (2023). “Neuroprotective Effects of Blueberries through Inhibition on Cholinesterase, Tyrosinase, Cyclooxygenase-2, and Amyloidogenesis.” Nutraceuticals 3, 39-57. https://doi.org/10.3390/nutraceuticals3010004.