بررسی ویژگی‌های آنزیم‌های آلفا آمیلاز و گلوکوزیداز غده گوارشی حلزون قهوه‌ای مرکبات در مجاورت روغن اسانسی آویشن، اسطوخدوس و مرزنگوش

یکی از آفات مهم باغ‌های مرکبات شمال ایران حلزون قهوه‌ای می‌باشد. امروزه به دلیل پیدایش مقاومت آفت‌ها به سموم شیمیایی و اثرات زیان‌بار و مخرب آنها بر سلامت و محیط زیست بشر، یکی از راه‌های کنترل آنها ارزیابی میزان اثر بازدارنده‌ها بر آنزیم‌های گوارشی، آلفا آمیلاز و آلفا گلوکوزیداز به نظر می‌رسد. هدف از این مطالعه، اندازه‌گیری اثر اسانس آویشن، اسطوخدوس و مرزنگوش از خانواده نعنائیان، برای مهار این دو آنزیم می‌باشد. ابتدا غده گوارشی حلزون قهوه‌ای در حالت گرسنگی جداسازی و از آن عصاره آنزیمی به روش‌های هموژنیزاسیون و سانتریفیوژ در دمای چهار درجه سانتی‌گراد تهیه شد. پارامترهای سینتیکی و فعالیت آلفا آمیلاز و آلفا گلوکوزیداز در عصاره آنزیمی اندازه‌گیری شد. اسانس‌گیری از پودر خشک هر سه گیاه به روش کلونجر انجام شد. فعالیت آلفا آمیلاز و آلفا گلوکوزیداز در مجاورت اسانس هر یک از گیاهان در غلظت‌های مختلف اندازه‌گیری شد. میزان پروتئین موجود در عصاره آنزیمی استخراج شده 0.2 میلی‌گرم در میلی‌لیتر به‌دست آمد. مقادیر km و vmax برای آلفا آمیلاز به‌ترتیب 0.13 میلی‌گرم در میلی‌لیتر و 0.062 میلی‌گرم در میلی‌لیتر بر دقیقه اندازه‌گیری شد. مقادیر ic50 برای آلفا آمیلاز در مجاورت اسانس‌های آویشن، اسطوخدوس و مرزنگوش به‌ترتیب 2.462 ، 0.2816 و 0.114 میلی‌گرم بر میلی‌لیتر و برای آنزیم آلفا گلوکوزیداز ic50 به‌ترتیب 0.741 ، 2.036 و 1.668 میلی‌گرم بر میلی‌لیتر به‌دست آمد. نتایج به‌دست آمده نشان داد که اسانس روغنی آویشن، اسطوخدوس و مرزنگوش می‌توانند آنزیم‌های آلفا آمیلاز و آلفا گلوکوزیداز جداسازی شده از حلزون را مهار نموده و به عنوان منابع طبیعی، کم هزینه و قابل دسترس برای کاهش خسارت به مرکبات مورد توجه قرار گیرند.

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.

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.