Investigating the Characteristics of α-Amylase and Glucosidase Enzymes in the Digestive Gland of Brown Citrus Snail in the Vicinity of Essential Oils of Thymus vulgaris L., Lavandula angustifolia Mill. and Origanum vulgare L.

Document Type : Research paper-Persian

Authors

1 M.Sc. of Biochemistry, Department of Molecular and Cell Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Iran

2 Associate Professor, Department of Molecular and Cell Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Iran

Abstract

Background and Objectives: The brown snail is a major pest affecting citrus orchards in Iran. Using chemical pesticides can contaminate the air, water, and nearby crops. Due to the emergence of pest resistance to chemical toxins and their harmful and destructive effects on human health and the environment, one of the methods to control snails is to evaluate the activity and inhibition of their digestive enzymes. α-‌amylase and α-‌glucosidase enzymes are key enzymes in the digestive system, and their inhibition is crucial in biotechnology. Using natural sources as inhibitors for these two enzymes is a new goal due to fewer side effects than chemical inhibitors. The purpose of this study was to use the essential oils of thyme (Thymus vulgaris L.), lavender (Lavandula angustifolia Mill.), and marjoram (Origanum vulgare L.) plants from the mint family to inhibit these two enzymes of the digestive gland of the brown snail.

Materials and Methods: In this study, α -amylase and α -glucosidase enzymes were extracted from brown citrus snails. The digestive glands were isolated from snails in starvation conditions. The enzyme extract was prepared after homogenization and centrifugation methods at 4 °C. Kinetic parameters and activity of α-amylase and α-glucosidase measured in the enzyme extract were measured. The total amount of proteins in the crude extract was measured by the Bradford method. The essential oil was prepared using a Clevenger apparatus. The activities and kinetic parameters of α-amylase and α-glucosidase enzymes in crude extract were measured in the presence of different concentrations of the essential oils of T. vulgaris, L. angustifolia and O. vulgare. α-amylase enzyme activity was measured according to Bernfeld's method at a fixed concentration of the starch solution, essential oils (0.1-6 mg/ml) and 250 µl of the enzyme extract. The total volume was 2.5 ml in each experiment. α-glucosidase enzyme activity was determined based on the method of Siegentaler and Low with a slight change. In each experiment, different amounts of the essential oils (0.25-5 mg/ml) were incubated with the enzyme extract (15 µl) and para-nitrophenyl alpha-di-glucopyranoside (20 µl). The total volume was 200 µl.

Results: The amount of protein in the crude enzyme extract was 0.2 mg/ml. The α-amylase activity was obtained at 0.278 U/ml, and the specific activity was 1.39 U/mg. The Km and Vmax values for α-amylase were measured as 0.13 mg/ml and 0.062 mg/ml/min, respectively. The α-glucosidase activity was obtained at 0.4 U/ml, and the specific activity was 2 U/mg. The IC50 values for the α-amylase in the presence of essential oils of T. vulgaris, and L. angustifolia. and O. vulgare were obtained at 2.462, 0.2816, and 0.114 mg/ml, and for α-glucosidase was obtained at 0.741, 2.036, and 1.668/ml, respectively. The GC-MS analysis found that the main constituents of the essential oils of T. vulgaris, L. angustifolia and O. vulgare were Carvacrol, D-Camphor, and Terpinene-4-ol, respectively. The essential oil of O. vulgare acted as an α-amylase inhibitor, and the thyme essential oil acted as an α-glucosidase inhibitor. Our experiments showed that the essential oil of L. angustifolia competitively inhibited the α-amylase. On the other hand, O. vulgare essential oil can act as an effective competitive inhibitor for the α-glucosidase. Therefore, compounds of D-Camphor and Terpineol, which are the main components of L. angustifolia and O. vulgare essential oils, are effective competitive inhibitors for α-amylase and α-glucosidase of the snail.

Discussion: Citrus snail is one of the most significant pests of agricultural products, which damages all kinds of citrus cultivars in gardens and nurseries by feeding on leaves, stems, fruits, and tree bark. Iran's rich pharmaceutical plant flora contains diverse compounds, including volatile ones like essential oils, which can be explored for their medicinal, insecticidal, fungicidal, and other properties. According to the literature, the snails have more than 20 types of carbohydrases. α-amylase and glucosidase are key enzymes in carbohydrate digestion and metabolism in insects that hydrolyze starch and glycogen. Our results showed that essential oils of T. vulgaris, L. angustifolia and O. vulgare or their active components can be proposed as environmentally friendly, low-cost, and accessible sources for the production of effective inhibitors against α-amylase and α-glucosidase of brown citrus snail.

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References

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 © 2024 by the authors. Licensee SCU, Ahvaz, Iran. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0 license) (http://creativecommons.org/licenses/by-nc/4.0/.
Plant Protection (Scientific Journal of Agriculture)
Volume 47, Issue 3
October 2024
Pages 1-22

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