اثر غلظت‌های مختلف کیتوزان بر تولید اسیدهای فنلی در کشت سلول کاهوی موجدار (Lactuca undulate Ledeb)

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی دکتری فیزیولوژی گیاهی گروه زیست شناسی، دانشکده علوم، دانشگاه گلستان، گرگان، ایران

2 عضو هیات علمی -دانشگاه گلستان

3 استادیار گروه زیست شناسی، دانشکده علوم، دانشگاه گلستان، گرگان، ایران

چکیده

هدف: استفاده از الیسیتورها در کشت سلولی گیاهان یکی از مهم‎ترین روش‎ها جهت افزایش تولید  متابولیت‌های ثانویه است. کیتوزان بیوپلیمری است که از واحدهای D-گلوکزآمین ساخته شده و در دیواره سلولی قارچ‎ها و اسکلت خارجی بندپایان یافت می‎شود. القای پاسخ‎های دفاعی، افزایش فعالیت آنزیم‎های آنتی‎اکسیدان و مسیر فنیل‎پروپانوئیدها، تجمع ترکیبات فنلی و فلاوونوئیدها از جمله پاسخ‌های گیاهان به تیمار کیتوزان می­باشد. هدف از پژوهش حاضر بررسی اثر کیتوزان بعنوان یک الیسیتور در افزایش تولید اسیدهای فنلی در سوسپانسیون سلولی کاهوی موجدار (Lactuca undulateLedeb) است.
مواد و روش‌ها: ابتدا سوسپانسیون سلولی از کالوس‎های 45 روزه حاصل از قطعه جداکشت برگ کاهوی موجدار در محیط کشت MS 2/1 حاوی 1/0 و 1 میلی­گرم در لیتر 2,4-D و Kin تولید شد. سپس اثر غلظت‎های مختلف کیتوزان (0، 50، 100 و 200 میلی­گرم در لیتر) در بازه زمانی 24، 48 و 72 ساعت بر سوسپانسیون سلولی مورد بررسی قرار گرفت. بعد از برداشت نمونه‎ها، درصد زنده‎مانی، مقدار فنل و فلاوونوئید کل، شیکوریک اسید، کلروژنیک اسید، کافئیک اسید و پراکسیداسیون لیپیدها و فعالیت آنزیم فنیل­آلانین­آمونیالیاز (PAL) مورد بررسی قرار گرفت.
نتایج: داده­های حاضر نشان داد که غلظت کیتوزان و مدت زمان تیمار عوامل تعیین‌کننده در مقدار ترکیبات فنلی ازجمله شیکوریک اسید در سوسپانسیون سلولی کاهوی موجدار است. مقدار شیکوریک اسید در تیمار 50 میلی‎گرم در لیتر کیتوزان پس از 24 ساعت نسبت به تیمار شاهد 8/2 برابر افزایش نشان داد. بیشترین مقدار کلروژنیک اسید و کافئیک اسید پس از 24 و 48 ساعت تیمار با غلظت 200 میلی­گرم در لیتر کیتوزان بدست آمد. بعلاوه نتایج حاضر بیانگر اثر تیمار کیتوزان بر افزایش فنل و فلاوونوئید کل همراه با افزایش فعالیت آنزیم PAL بوده است. افزایش مقدار پراکسیداسیون لیپیدها و کاهش درصد زنده‎مانی سلول‎ها در غلظت‎های بالای کیتوزان نشان‎دهنده اثر منفی آن بر فعالیت سلول‏ها است.
نتیجه‌گیری: نتایج حاضر نشان داد کیتوزان در غلظت‎های پایین سبب افزایش مقدار شیکوریک اسید در سوسپانسیون سلولی کاهوی موجدار شده که از آن می‎توان در صنایع داروسازی به عنوان روشی نوین در تولید شیکوریک اسید و مشتقات آن استفاده کرد.

کلیدواژه‌ها


عنوان مقاله [English]

The effect of different concentrations of chitosan on the production of phenolic acids in cell culture of lactuca undulate ledeb

نویسندگان [English]

  • Morteza Mofid Bojnoordi 1
  • Mahnaz Aghdasi 2
  • Mohammad Fatemi 3
1 PhD Candidate, Department of Biology, Faculty of Science, Golestan University, Gorgan, Iran.
2 academic staff- golestan university
3 Assistant Professor, Department of Biology, Faculty of Science, Golestan University, Gorgan, Iran.
چکیده [English]

Objective
One of the most important methods for increasing the production of secondary metabolites is the use of elicitors in plant cell culture. A biopolymer made from D-glucosamine units, chitosan can be found in the cell walls of fungi and in the exoskeletons of arthropods. Plants respond to chitosan treatment by generating defense responses, increasing antioxidant enzyme activity, accumulating phenolic compounds, and releasing flavonoids. In the current study, chitosan was used as an elicitor to induce the production of phenolic acids in Lactuca undulata cells suspended in liquid medium.
 
Materials and methods
First, cell suspension culture was prepared from 45 days old callus derived from leaf explants of Lactuca undulata on ½ MS medium supplemented with 0.1 and 1 mgL-1 2,4-D and Kin. The effect of different concentrations of chitosan (0, 50, 100 and 200 mgL-1) on cell suspension was evaluated during 24, 48 and 72 hours. After harvesting samples, the percentage of cell viability, total phenols and flavonoids, chicoric acid, chlorogenic acid and caffeic acid contents, as well as phenylalanine-ammonia lyase (PAL) activity and lipid peroxidation were measured.
Results
In the present study, we found that the concentration and duration of chitosan treatment affect the production of phenolic compounds (including chicoric acid) in Lactuca undulata cell suspension culture. After 24 hours of treatment with 50 mgL-1 chitosan, chicoric acid levels had increased 2.8-fold compared to the control. After 24 and 48 hours of treatment with 200 mgL-1 chitosan, the highest levels of chlorogenic and caffeic acids were observed. Furthermore, the present study found the chitosan treatment resulted in an increase in the amount of phenol and total flavonoids as well as an increase in PAL enzyme activity. Chitosan induces lipid peroxidation and reduces cell viability in high concentrations, indicating a negative effect.
 
Conclusions
The present study found that low concentrations of chitosan could induce chicoric acid production in Lactuca undulata cell suspension cultures, which can be utilized in the pharmaceutical industry as a new method in the production of chicoric acid and its derivatives.

کلیدواژه‌ها [English]

  • Cell suspension culture
  • cichoric acid
  • Lactuca undulata
  • PAL
  • total phenol
 
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