Inhibitory Effect Of Silkworm Extract On Alpha-Glucosidase Activity And PostprAndial Blood Glucose In Mice

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PHARMACEUTICAL ENGINEERING®
The Official Magazine of ISPE November/December 2011, Vol. 31 No. 6

Diabetes Research

This article was developed from the presentation by a finalist in the ISPE 2010 Undergraduate International Student Poster Competition.

Inhibitory Effect of Silkworm Extract on Alpha-Glucosidase Activity and Postprandial Blood Glucose in Mice
byJuan Cueva, Patricio Castillo, Giovanna Allara-Salice, and Angel Guevara

iabetes mellitus type 2 is a metabolic disorder with high percentages of morbidity and mortality.1 Due to an asymptomatic pre-diabetic phase, 40% of patients are not aware of their condition until they have reached an advanced stage. However, in some cases, prevention and control of type 2 diabetes is possible with changeof habits and/ or therapeutic treatment.1 Long-term hyperglycemia has a tight relationship with type 2 diabetes development. Thus, patients on a pre-diabetic state have a higher risk to develop this condition and cardiovascular disease.2 Throughout time, hyperglycemia contributes to insulin resistance; the risk of diabetes increases annually from 3.6 to 8.7% on Impaired Glucose Tolerance (IGT)patients.3 Similarly, coronary artery disease lipid and non-lipid risk factors are associated with a pre-diabetic state whether a diabetic condition is shown or not in the patient.4 1-deoxynojirimycin (DNJ), a poly-hydroxylated alkaloid isolated from mulberry leaves5 and silkworm,6 has been found to be a potent inhibitor of intestinal alpha-glycosidases.5,7 However, there are only few sources ofevidence that address the potential of silkworm as a blood glucose-lowering product on diabetic patients;8,9 moreover, there is limited investigation related to the specific enzyme inhibition mechanism. In the present study, an ethanol extract from silkworm larvae was prepared and tested in vitro for alpha-glucosidase activity inhibition. Furthermore, postprandial antihyperglycemic effect was examinedin mice by using oral doses of SLE. Analysis of the results shows that SLE or other food products derived from silkworm larvae could contribute positively to diabetes
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D

Introduction

mellitus prevention if used as an alternative dietary supplement.

Materials

Experimental Procedures

Alpha-glucosidase (EC 3.2.1.20) from baker’s yeast and a Glucose (HK) Assay KitGAHK-20 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Maltose monohydrate was purchased from Himedia Laboratories (Mumbai, India). Silkworm larvae were provided by Instituto Agropecuario Superior Andino IASA II (Escuela Politécnica del Ejército, Santo Domingo, Ecuador).

Preparation of Silkworm Larvae Extract
Third-day, fifth-instar Bombyx mori larvae were collected on asepticconditions. An appropriate amount of larvae was homogenized with 4°C water and extracted with 50% ethanol for 72 hours. Then, the extract was filtered and centrifuged to separate from solids. Concentration was performed on a rotavapor at 50°C until a volume of 50% was reduced from the original value. The concentrated extract was then lyophilized (-46°C and 145E-3 mbar for 48 hours) and frozen at -20°Cuntil further use.

Inhibition Assay
Various concentrations of SLE (0.02-14.00 mg/ml) were premixed with maltose solution (50 mM) in phosphate buffer + EDTA (pH 7.) and incubated at 37°C for five minutes. The reaction was started with the addition of alpha-glucosidase (0.05 mg/ml), carried out at 37°C, and stopped with boiling water. Alphaglucosidase activity was determined by measuring theconcentration of glucose released from 1

NovembeR/DecembeR 2011 PHARMACEUTICAL ENGINEERING Online Exclusive

Diabetes Research
tion + SLE [0.4g/kg]) by using glucometer Accu-Check Active (Roche® Diagnostics, Mannheim, Germany) and by following blood collection procedures described by Hoff.11 Samples were collected before the oral treatment administration and after 30, 90, 60, and 150 minutes....