سنتز سبز نانوذرات نقره با استفاده از عصاره برگ علف هرز تاج خروس و بررسي اثر آنتي‌باكتريايي

Green Synthesis of Silver Nanoparticles Using Amarantus retroflexus and Its Antibacterial Effect

عصاره گیاهان به عنوان یك كاهنده گیاهی برای تولید سبز، آسان، غیرسمی و دوستدار محیط زیست نانوذرات نقره مورد استفاده قرار گرفته است. در این مطالعه سنتز زیستی نانوذرات نقره با استفاده از عصاره برگ علف هرز تاج خروس ریشه قرمز Amaranthus retroflexus به عنوان كاهنده و پایدار كننده در سنتز نانوذرات نقره مورد استفاده قرار گرفت. تأثیر پارامترهای مختلف از جمله اسیدیته مخلوط واكنش، زمان تماس، نسبت عصاره به محلول نیترات نقره بر روی سنتز نانوذرات نقره مورد مطالعه قرار گرفت. تشكیل نانوذرات در داخل محلول نیترات نقره در ابتدا با استفاده از طیف‌نگاری در دامنه طول موج 200 تا 800 نانومتر توسط دستگاه طیف‌نگار UV–vis مورد تأیید قرار گرفت. مورفولوژی نانوذرات تشكیل شده با استفاده از میكروسكوپ الكترونی عبوری مورد مطالعه قرار گرفت و نتایج نشان داد شكل نانوذرات كروی بوده و مونودیسپرس بوده و دامنه اندازه ذرات آن بین 2 تا 30 نانومتر بود. ساختار كریستالی نانوذرات به روش XRD مورد بررسی قرار گرفت و گروه‌های عاملی مسئول كاهش و پایدارسازی نانوذرات نقره با استفاده از طیف‌سنجی FT-IR مورد ارزیابی قرار گرفت. حداكثر سنتز نانوذرات در نسبت حجمی (v/v) عصاره به محلول نیترات نقره 1/0، 9=pH و مدت زمان واكنش 240 دقیقه مشاهده شد. بعد از تولید نانوذرات نقره زیستی، خاصیت ضدباكتریایی آن بر روی برخی پاتوژن‌های مقاوم انسانی؛ E. coli(Migula 1895) و Pseudomonas aeruginosa (Schröter 1872) و همچنین پاتوژن‌های مقاوم گیاهی Pseudomonas syringae(Van Hall, 1904) و Xanthomonas oryzae(Ishiyama 1922) مورد مطالعه قرار گرفت و با باكتری¬كش سیپروفلوكسازین مقایسه شد. نتایج مطالعه حاضر نشان داد كه گیاه تاج خروس گزینه مناسبی برای تولید نانوذرات نقره بوده و نانوذرات تولید شده به این روش دارای خواص آنتی‌باكتریایی هستند. نانوذرات تولید شده دارای شكل كروی بوده و میانگین اندازه آنها 74/10 نانومتر بود كه با نتایج حاصل از آنالیز XRD همخوانی داشت. نتایج FTIR نشان داد كه آلكالوئیدها و سایر تركیبات ثانویه گیاه مسئول اصلی سنتز نانوذرات هستند. پارامترهای مختلف مورد مطالعه بر سنتز نانوذرات تأثیرگذار بودند. محیط‌های قلیایی برای سنتز نانوذرات مناسب تر بوده و درصد عصاره به نیترات نقره 1/0 بهترین كارایی را داشت. تأثیر زمان بعد از 1 ساعت اختلاف معناداری نداشت.

Introduction: Silver nanoparticles are important materials that have been studied extensively. They have unique physical, chemical and biological properties such as potential antibacterial activity. They can be synthesized by several physical, chemical methods. However, these methods are not environmentally friendly. Therefore, it is desirable to develop an eco-friendly method such as utilizing the biological species plant extracts for the synthesis of various environmental friendly metal nanoparticles. In addition, the integration of green chemistry principles into nanotechnology is essential. The biomolecules found in plants induce the reduction of Ag+ ions from silver nitrate to AgNPs. The process of reduction is extra cellular and fast leading to the development of easy biosynthesis of silver nanoparticle. The green synthesis of AgNPs using plants [6]–[11] have been before reported .Plant extract have been used as a natural reductant to produce green, non-toxic and environmentally friendly silver Nano particles. Materials and Methods: to biosynthesis silver nanoparticles, Amaranthus retroflexus leaf extract was used as reductant and stabilizer agent. The effect of various parameters such as pH of the reaction solution, contact time, ratio of extract to silver nitrate solution on the synthesis of silver nanoparticles was studied. For reduction of silver ions, 1 mM silver nitrate was added to the weed leaves extracts with different ratio (v/v) of extract to silver nitrate with constant stirring under room temperature. The final nano-colloidal solution was subjected to repeat centrifugation (thrice) to get rid of any uninteracted biological molecules at 12,000 rpm for 15 min and the pellet were dried in vacuum oven. Formation of nanoparticles in the silver nitrate solution primarily was detected using UV-vis spectrometer (wavelength range 200 to 800 nm). The morphology of the nanoparticles studied using transmission electron microscopy. The crystalline structure of nanoparticles was studied by XRD and functional groups responsible for the reduction and stabilization of silver nanoparticles was evaluated using FT-IR spectroscopy. Pseudomonas aeruginosa was used as a sample bacteria for antibacterial studies. The antibacterial activity of AgNPs was investigated against gram negative drug resistant P. aeruginosa. For this reason, various concentrations of AgNPs from 400 to 1.56 µg/ml were incubated with cells of pathogenic bacteria in liquid medium. Growth of bacterial cells at the presence of AgNPs was compared with the growth of bacterial cells (positive control) in the absence of NPs. percentage of bacterial growth inhibition was evaluated at different AgNPs concentrations and MIC was determined against P. aeruginosa. Results: The results of TEM showed that the nanoparticles were spherical and monodispersed with the particle size between 2 and 30 nm. The maximum nanoparticles synthesis occurs in silver nitrate to extract ratio (v / v) of 0.1, pH 9 and reaction time of 240 minutes. The FTIR result indicated the involvement of amides, carboxyl, amino groups and amino acid residues present in leaf extract in the NP synthesis. The XRD confirmed the structure of silver as a face-centered cubic structure [14]. Therefore, the XRD data clearly demonstrated the presence and crystal structure of the silver in the A. retroflexus leaf extract. Growth of bacterial cells was completely inhibited at the 200 µg/ml of AgNPs considered as the MIC. The antibacterial activity of AgNPs compared favorably with standard antibiotics including ciprofloxacin and cephtazidim. MIC of ciprofloxacin toward tested pathogen were 0.125 µg/ml which shows stronger antimicrobial activity in comparison with AgNPs. In contrast, only 50% of bacterial growth inhibition was observed at the 256 µg/ml of cephtazidim. Thus, the antibacterial activity of AgNPs was stronger than cephtazidim against P. aeruginosa. Leaves extract alone did not display any antimicrobial activity against tested microorganism. Conclusion: AgNPs were successfully synthesized using Amaranthus retroflexus leaf extract as a reducing and capping agents. The structural, morphological and elemental studies of biologically synthesized AgNPs were characterized by UV-vis spectrometery, atomic absorption, XRD, FTIR, and TEM. The presence of AgNPs (200 µg/ml) was significantly reduced the cell viability of P. aeruginosa compared to control after 24 h incubation period. Biological synthesized silver nanoparticles could be of immense use in the medical field for their efficient antimicrobial function.