افزایش تولید آلکالوئیدها و ترکیبات فلاونوئیدی کالوس نرگس شهلا (.narcissus tazetta l) تحت تیمار نانوذرات اکسید آهن (-nps fe3o4) و پیش‌ماده‌های ترانس سینامیک‌اسید و l- فنیل آلانین

خواص دارویی گیاه نرگس به علّت وجود آلکالوئیدهای معروف به آلکالوئیدهای آماریلیداسه به ویژه در پیازهای آن است. تنش اکسیداتیو ناشی از حضور نانوذرات، تولید متابولیت‌های ثانوی را با تولید گونه‌های فعال اکسیژن (ros) و فعال‌کردن مسیرهای علامت‌دهی تحریک می‌کند. استفاده از پیش‌ما‌ده‌های خاص مسیر بیوسنتزی آلکالوئیدها می‌تواند در افزایش تولید این ترکیبات موثر باشد. در این پژوهش، اثر نانوذرات اکسید آهن (fe3o4-nps) و پیش‌ماده‌های ترانس سینامیک‌اسید (tca) و l- فنیل‌آلانین (l-phe) بر تغییر برخی فاکتورهای بیوشیمیایی کالوس‌های narcissus tazetta l. به ویژه محتوای آلکالوئیدها بررسی شد. نتایج نشان داد هنگامی که کالوس‌های القاء شده در محیط کشت‌ موراشیگ- اسکوگ (ms) حاوی هورمون‌های 2,4-d و bap با fe3o4-nps تیمار شدند، متابولیت‌های ثانوی مانند آلکالوئیدها، فلاونوئیدها و ترکیبات فنولی به‌طور معنی‌داری افزایش یافت. همچنین، فعالیت آنزیم‌های پاد اکسایشی سوپر اکسید ‌دیسموتاز (sod)، کاتالاز (cat) و پراکسیداز (pod)، آنزیم‌های فنیل‌آلانین آمونیالیاز (pal) و تیروزین آمونیالیاز (tal) تحت تاثیر تیمارهای  fe3o4-nps به تنهایی و در ترکیب با هریک از دو پیش ماده‌ی ذکر شده افزایش نشان داد. ارزیابی تغییر فاکتورهای مورد بررسی نشان داد کاربرد tca و l-phe متابولیسم گیاه را در جهت بهبود تولید متابولیت‌های ثانوی تغییر دادند. برای مثال در 15 روز تیمار با فنیل‌آلانین، فعالیت آنزیم‌های sod، cat و pod به ترتیب 57.21، 77.72 و 78.5 درصد نسبت به شاهد افزایش نشان داد. این امر به ویژه با کاربرد همزمان fe3o4-nps و پیش‌ماده‌ها شاخص‌تر بود. با افزودن این پیش‌ماده‌ها به محیط کشت، محتوای h2o2 تحت تیمار tca افزایش و تحت تیمار l-phe کاهش یافت. افزایش فعالیت آنزیم‌های پاد‌اکسایشی ناشی از تیمارهای نانوذرات همراه با اثر تغذیه‌ای پیش‌سازهای tca و l-phe، ضمن فعال کردن مسیر علامت‌دهی پاسخ‌های پاد‌اکسایشی، فعالیت آنزیم‌های درگیر تولید برخی متابولیت‌های ثانوی از جمله مسیر بیوسنتزی آلکالوئیدها و ترکیبات فنولی را القا کرد و محتوای این متابولیت ها را در کالوس افزایش داد. بهینه‌سازی و افزایش تولید آلکالوئیدهای آماریلیداسه با توجه به خواص ضد آلزایمری، ضد ویروسی، ضد سرطانی و پاداکسایشی از لحاظ تجاری و پزشکی حائز اهمیت است.

improvement of production of alkaloid and flavonoid compounds in narcissus tazetta l. callus under iron oxide nanoparticles (fe3o4-nps) and trans-cinnamic acid and l-phenylalanine treatments

the medicinal properties of the narcissus genus are due to the presence of alkaloids known as amaryllidaceae alkaloids (aa). the presence of nanoparticles causes oxidative stress and stimulates production of secondary metabolites. useing specific precursors in aa biosynthetic pathways can be effective on cumulative production of these compounds. in this study, the effect of iron oxide nanoparticles (fe3o4-nps), trans-cinnamic acid (tca), and l-phenylalanine (l-phe) as precursors has been investigated on changes in some biochemical parameters of narcissus tazetta l. callus, especially alkaloid content. treatment of calli generated from bulb explants in morashig-skoog (ms) media containing 2,4-d and bap and fe3o4-nps, increased alkaloids, flavonoids, and phenolic compounds. also, the activity of antioxidant enzymes (sod, cat and pod), and activity of phenylalanine ammonia-lyase (pal) and tyrosine ammonia-lyase (tal) increased under fe3o4-nps treatments alone and in combination with tca or l-phe precursors. the evaluation of the studied parameter changes showed that both tca and l-phe treatment can change the plant’s metabolism to improve the production of secondary metabolites. this was especially evident with the simultaneous application of fe3o4-nps and these precursors. application of precursors in the culture medium increased h2o2 content under tca treatment and decreased it under l-phe treatment. increased activity of the antioxidant enzymes caused by nanoparticle treatments along with the nutritional effect of tca and l-phe precursors, progressed signaling pathway of antioxidant responses by affecting the activity of biosynthetic enzymes and increases production of some secondary metabolites including alkaloids and phenolic compounds. considering various properties of amaryllidaceaea alkaloids, optimizing, and increasing the production of these alkaloids is important from a commercial and medical point of view. introductionthe incomparable feature of narcissus plants, especially in bulbs, is the production of amaryllidaceae alkaloids with worthwhile medicinal properties. these alkaloids have a wide range of biological activities, such as anti-alzheimer properties, antiviral and antitumor activities and antioxidant and anticancer properties. the content of these alkaloids in different narcissus species is small, and the chemical synthesis of these compounds is challenging due to their complex structure. therefore, the extraction and industrial production of these alkaloids on a large scale is not economical. meanwhile, the use of alternative and low-cost methods to increase these valuable medicinal compounds has been considered. among these methods, tissue culture and use of elicitors can help to this goal. iron nanoparticles can be mentioned among these stimulants. fe3o4-nps are one of the most important forms of iron oxide nanoparticles that are widely used in biomedicine and agriculture researchs as elicitor. these nanoparticles cause various biochemical and physiological changes in plants. also, some compounds such as phenylalanine and tyrosine, are specific precursors in the biosynthetic pathway. the effect of exogenous phenylalanine and tyrosine was studied on the secondary metabolism of wheat (feduraev et al., 2020). the results showed that along with the increase in phenylalanine and tyrosine, the content of phenolic compounds and the activity of pal, tal, and pod increased. also, marconi et al. (2007) were investigated the effect of exogenous trans-cinnamic acid on the changes of alkaloid content in datura (brugmansia candida pres.). cinnamic acid harmed plant growth and caused an increase in plant alkaloids, especially scopolamine.aim of this study was investigation on (i) the effects of fe3o4-nps, tca and lphe on the changes in phenolic, flavonoid and alkaloid contents and (ii) investigation of the effect of oxidative stress due to nanoparticle treatment on the production of secondary metabolites. materials and methodsthe prepared bulbs of narcissus tazetta l. var. shahla was disinfected and transferred to the ms culture media containing 1 mg/l of 2,4-d and bap to induce callus. iron oxide nanoparticles (fe3o4 -nps) were purchased from mashhad nanosadra company and were homogenized for 30 minutes with ultrasonic waves (60 w power) to prepare a mother solution of 500 mg/l. also, two precursors of trans-cinnamic acid and l-phenylalanine were prepared and sterilized at a concentration of 200 mg/l. thirty-five days old calluses were transferred to 1/2 ms liquid medium containing nanoparticles (0 and 500 mg/l) and precursors (0 and 200 mg/l) alone or together and put on a shaker with a speed of 100 rpm at 25 °c. the calli were harvested at two-time stages, 2 and 15 days after treatments, and stored in a freezer at -80°c for biochemical tests. total phenolic compounds, flavonoids, alkaloid, and hydrogen peroxide, mda content, enzyme activities of sod, pod, cat, tal, and pal were assayed. results and discussionthe finding of this study revealed that fe3o4-nps, tca, and l-phe improved productions of the secondary metabolites in narcissus tazetta l. callus. the results showed that fe3o4-nps and l-phe decreased h2o2 content, but tca increased hydrogen peroxide content in calli. treatments by fe3o4-nps alone and combined with tca or l-phe increased the activity of antioxidant enzymes including cat, pod, and sod. the content of total phenolic, flavonoid, and alkaloid compounds in n. tazatta calli increased under the treatments of fe3o4-nps and both precursors. the activity of pal and tal enzymes increased significantly under all the treatments. iron oxide nanoparticles, as a compound that provides a vital nutrient, are involved in several metabolic processes in plants (hu et al., 2017). phenylalanine is a precursor of many secondary metabolites such as phenolic acids, flavonoids, anthocyanins, lignin, and alkaloids. trans-cinnamic acid, as the first chemical substance which produce in the phenylpropanoids pathway, is the precursor of many hydroxycinnamic acid derivatives and plays a key role in the formation of other complex phenolic compounds (swanson, 2003). thus, treatment of the calli by fe3o4-nps, tca, and l-phe can be active metabolism of the n. tazetta to ward production of the important phenolic and alkaloid compounds. conclusiontreatment of the n. tazetta calli with fe3o4-nps, tca, and l-phe increased their ability to produce secondary metabolites by increasing h2o2 and changes in mda levels. fe3o4-nps act through signaling pathways of ros and induction of antioxidant enzyme activities. on the other hand, these treatments increased pal and tal activities and improved phenolic and alkaloid biosynthetic pathways.