Microsoft Word - 11199 NSB Sharma 2023.03.06.docx Received: 02 Feb 2022. Received in revised form: 14 Dec 2022. Accepted: 06 Mar 2023. Published online: 16 Mar 2023. From Volume 13, Issue 1, 2021, Notulae Scientia Biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. The journal will continue to appear quarterly, as before, with four annual numbers. SHSTSHSTSHSTSHST Horticulture and ForestryHorticulture and ForestryHorticulture and ForestryHorticulture and Forestry Society of TransylvaniaSociety of TransylvaniaSociety of TransylvaniaSociety of Transylvania Sharma B and Alam A (2023) Notulae Scientia BiologicaeNotulae Scientia BiologicaeNotulae Scientia BiologicaeNotulae Scientia Biologicae Volume 15, Issue 1, Article number 11199 DOI:10.15835/nsb15111199 Research ArticleResearch ArticleResearch ArticleResearch Article.... NSBNSBNSBNSB Notulae Scientia Notulae Scientia Notulae Scientia Notulae Scientia BiologicaeBiologicaeBiologicaeBiologicae Biogenesis of silver nanoparticles from the shoot extract of Biogenesis of silver nanoparticles from the shoot extract of Biogenesis of silver nanoparticles from the shoot extract of Biogenesis of silver nanoparticles from the shoot extract of Delonix Delonix Delonix Delonix regiaregiaregiaregia its characterization (UVits characterization (UVits characterization (UVits characterization (UV––––Vis spectroscopy and SEM) and Vis spectroscopy and SEM) and Vis spectroscopy and SEM) and Vis spectroscopy and SEM) and evaluation for antimicrobial potentialevaluation for antimicrobial potentialevaluation for antimicrobial potentialevaluation for antimicrobial potential Bhawana SHARMA, Afroz ALAM* Banasthali Vidyapith, Department of Bioscience and Biotechnology, Rajasthan-304022 India; bhawana.sharma1230@gmail.com; afrozalamsafvi@gmail.com (*corresponding author) AbstractAbstractAbstractAbstract The current research work explores the production of silver nanoparticles using aqueous extracts of Delonix regia (Boj. ex Hook.) Raf. (Angiosperms; Fabaceae) shoots for the bioreduction of Ag metal and its antimicrobial activity. Fourier transform infrared spectroscopy (FTIR), Zeta potential, ultraviolet-visible spectroscopy, scanning electron microscopy (SEM), and X-ray diffraction (XRD) have been used to evaluate the produced silver nanoparticles (AgNPs). Both antibacterial and antifungal activity were examined against bacterial and fungal pathogens, viz., Escherichia coli and Bacillus subtilis, and fungal strains, viz., Fusarium oxysporum and Aspergillus niger. The presence of silver nanoparticles was observed by the color change, i.e., from pale yellow to dark brown. The zeta potential observed for the produced nanoparticle is -18mv. The SEM and XRD revealed the size of synthesized AgNPs, i.e., 35nm and SEM size lies in the range of 40-60 nm. UV- visible absorption spectra were found at wavelength 425 nm. The synthesized nanoparticles are cost-efficient and could be an alternative procedure for the peculiar production of nanoparticles and also act as potential antimicrobial agents. KeywKeywKeywKeywords:ords:ords:ords: AgNPs; antimicrobial activity; Delonix regia; FTIR; SEM; XRD IntroductionIntroductionIntroductionIntroduction Silver nanoparticles (AgNPs) are gaining popularity in a variety of fields, including medicine, food, industrial applications, and healthcare. They are typically accomplished through a variety of chemical and physical methods such as a specific rod position, laser ablation, and pyrolysis, all of which appear to be extremely dangerous and costly (Singh et al., 2010). Biogenic silver nanoparticle (AgNPs) synthesis is efficient, sustainable, cost-effective, and ecofriendly, and has proven to be one of the viable and novel mechanisms for the peculiar production of silver nanoparticles (AgNPs). In recent years, various metals and materials have been involved in the production of nanoparticles from algae, fungi, bacteria, viruses, and several plants (Valli and Geetha, 2016). The determination of biological activity of AgNPs is done under various parameters, which include particle size, shape, and composition, which are used for determining cytotoxicity. Due to the attractive physicochemical properties of silver nanoparticles, they have great potential in the fields of medicine and https://www.notulaebiologicae.ro/index.php/nsb/index Sharma B and Alam A (2023). Not Sci Biol 15(1):11199 2 biology (Akter et al., 2018). Metallic nanoparticles have exhibited enhanced antioxidant, antimicrobial, and numerous other properties due to their small size and shape, high surface area, and distinct characteristics (Mubarak Ali et al., 2011; Dikshit et al., 2021). Delonix regia (Boj. ex Hook.) Raf. (Family Fabaceae), is a large, medium-sized ornamental tree with fern- like, bipinnately compound leaves. It is commonly known as Royal Poinciana, Flamboyant (English), and Gulmohar (Hindi). It is mainly found in Madagascar, Africa, Northern Australia, and India as a semi- deciduous tree (Latif et al., 2019). Distinct parts of the D. regia plant, like leaves, roots, bark, and flowers, are used for the treatment of various ailments, like fever, bronchitis, anemia, pneumonia, diabetes, and inflammation. It exhibits various physiologically active chemical components such as lupeol, coumarin, carotene, lycopene, sterols, gallic acids, quercetin, etc. D. regia is generally used for treating fungal and bacterial infections, inflammation, and liver diseases (Siddiquee et al., 2020). Therefore, the aim of this study was to analyze the production of environmentally compatible silver nanoparticles through D. regia shoot extract. Different techniques were used for the characterization of silver nanoparticles, such as UV, SEM, XRD, and their antimicrobial activity. Phytoconstituents of Delonix regia The plant consists of various pharmacologically active compounds like phenolic compounds that possess many anti-inflammatories, anti-ulcer, anti-microbial, anti-fungal, anti-bacterial, and anti-helmitic properties that are valuable or beneficial in a variety of health conditions (Bhorga and Kamble, 2019). Phytochemical screening results showed the presence of various bioactive constituents, like leaves that contain alkaloids, carbohydrates, and tannins; bark and shoots that contain terpenoids, flavonoids, tannins, alkaloids, and sterols; and flowers that contain saponins, steroids, carotenoids, and tannins. Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods Hi-media was used to make silver nitrate (AgNO3). Many other reagents, solvents, and materials used are of analytic grade. For the characterization of silver nanoparticles, both milli-Q water and double-distilled water were used. Collection and identification of plant extract The shoot of D. regia was collected from the campus of Banasthali Vidyapith (Tonk), Rajasthan, India. The authentication number of the reference specimen is BURI-1407/2022, and it was deposited in the herbarium of Banasthali University, Rajasthan, India (BURI). Preparation of D. regia shoot extract The collected shoots were rigorously washed with running tap water afterwards using milli-Q water. Before the pulverization process, the shoots were properly dried at room temperature for 10 days so that no moisture content was present. To prepare the shoot extract, 10 g of finely powdered shoot was mixed with 250 ml of deionized water and boiled at 60 °C for 30 min. Further, the resulting extract was cooled to room temperature before being filtered with Whatman No. 1 filter paper. For subsequent research, the extract which was properly filtered is retained at 4 °C (Das et al., 2017). Preparation of biogenic silver nanoparticles Following the bioreduction of silver nanoparticles (AgNPs), a 1 mM aqueous solution of AgNPs is produced. A 10 ml D. regia shoot extract was mixed with 90 ml of a 1 mM AgNO3 solution and incubated at Sharma B and Alam A (2023). Not Sci Biol 15(1):11199 3 28 °C for 24 hours. This bioreduction of AgNO3 can be visually apparent by the change in color pattern, i.e., from pale yellow to dark brown, as depicted in Figure 1. The confirmation of bioreduction was done by the UV visible spectra analysis pathway (Bharathi et al., 2018). FigureFigureFigureFigure 1. a1. a1. a1. a. Delonix regia shoot extract; b. b. b. b. Formation of silver nanoparticles Descriptive characterization of silver nanoparticles (AgNPs) Ultraviolet- Visible Spectrophotometer The synthesis of AgNPs was carried out by using a UV-visible spectrophotometer, noticing a distinctive surface plasmon resonance (SPR) peak at a scanning range of 200–800 nm and a speed of 200 nm/min. The dilution of the colored reaction samples was done with deionized water and scanned in ultra-violet visible spectra with a 1 cm quartz cuvette at 25 °C (Chouhan and Guleria, 2020). X-ray diffraction studies (XRD) The Bruker Eco D8 Advance X-pert PRO was used to figure out the X-ray diffraction of silver nanoparticles at 40 kV of voltage and a current strength of 20 mA (Mittal et al., 2012). Debye- Scherrer equation was used to calculate X-ray diffraction- D= Κλ/β cosϴ Where, D denotes crystal size Κ = shape factor (0.94) β = full width in radians at half maximum λ = denotes the X-ray wavelength (1.5418Ă) Particle size analyzer After 24–48 h of incubation, the particle size was measured using a Zetasizer (Santhoshkumar et al., 2011). Fourier transform infrared spectroscopy (FTIR) FTIR analysis was used to investigate the identification of functional groups on the surface of silver nanoparticles by a shoot extract of D. regia within an isolation distance of 4 cm (Reddy et al., 2019). Scanning Electron Microscopy (SEM) The morphology of D. regia stabilized nanoparticles was analyzed by scanning electron microscopy (Ahmed et al., 2016). Antimicrobial assay The antimicrobial activity of Delonix regia shoot extract and its AgNPs was explained by the disc diffusion method. Both Potato Dextrose Agar (PDA) and Nutrient Agar Media (NA) were used for the Sharma B and Alam A (2023). Not Sci Biol 15(1):11199 4 preparation of fungal and bacterial species. The AgNPs, AgNO3, and antibiotics were loaded into different filter paper discs. The discs were placed on nutrient agar media with bacterial culture and incubated for 12 h at 37 °C, whereas the discs were placed on potato dextrose agar with fungal culture and incubated for 24 hours at 28 °C. After 12 or 24 h, both bacterial and fungal cultures were checked, and the zone of inhibition was recorded (Rathi Sre et al., 2015). Results Results Results Results Characterization of AgNPs Ultra- violet visible spectroscopy After 24 h of observation, the ultraviolet-visible absorption spectra of colloidal silver nanoparticles (AgNPs) were shown. The peak intensity is found near 425 nm, indicating the formation of silver nanoparticles. The agitation of the Surface Plasmon Resonance (SPR) band in the U.V. visible region results in the color change from pale yellow to dark brown (aqueous solution), as depicted in Figure 2. Figure 2Figure 2Figure 2Figure 2. UV visible spectrophotometer of shoot extract of Delonix regia (AgNPs) X-ray diffraction (XRD) The XRD pattern of crystalline AgNPs is described in Figure 3. Sharp peaks of AgNPs were observed at 38.5°, 39.94°, 44.36°, 64.42°, and 77.44°. The calculation of average sizes of crystallized AgNPs was done using the Debye-Scherrer equation, and the size of AgNPs was observed to be 35.5 nm, which confirms the crystalline nature of synthesized silver nanoparticles (AgNPs). Sharma B and Alam A (2023). Not Sci Biol 15(1):11199 5 Figure 3Figure 3Figure 3Figure 3. X-ray diffraction pattern of AgNPs using Delonix regia shoot extract Particle size analyzer The sharp peak of the D. regia shoot extract was found to be 88.94 nm. The obtained results revealed that the diameter of AgNPs is 129.6 d (nm), as depicted in Figures 4 and 5. The zeta potential of D. regia shoot extract was investigated and found to be -18.7 mV. Figure 4. Figure 4. Figure 4. Figure 4. Particle size of silver nanoparticles (AgNPs)using Delonix regia Sharma B and Alam A (2023). Not Sci Biol 15(1):11199 6 Figure 5Figure 5Figure 5Figure 5. Zeta potential of AgNPs (Delonix regia shoot extract) Fourier- Transform Infrared Spectroscopy (FTIR) For the identification of the chemical bonds that are present in biogenic AgNPs, the FTIR tool is used. The spectra of D. regia shoot extract exhibit various absorption bands at 3367, 2920, 1630, and 1472 cm-1. The band is observed at 3367 cm1 due to OH- stretching vibrations. The different bands at 2920, 1630, and 1472 cm-1 indicate the existence of aromatic, carbonyl, and amide groups, as well as alkane groups (-C-H stretching). The minor bands at 787, 733, and 683 cm-1, which are attributes of aromatic phenols, indicate the presence of C-H out of plane bend regions as depicted in Figure 6. Figure 6Figure 6Figure 6Figure 6. Fourier transform infrared spectroscopy of AgNPs using Delonix regia shoot extract Scanning electron Microscopy (SEM) The investigation of the topology and size of the prepared samples was done through SEM. The results explained that the crystallized synthesis of silver nanoparticles exists in a rectangular shape, i.e., an average size of 40–60 nm, as depicted in Figure 7. Sharma B and Alam A (2023). Not Sci Biol 15(1):11199 7 Figure 7Figure 7Figure 7Figure 7. Scanning electron microscopy of synthesized AgNPs using Delonix regia shoot extract Antimicrobial activity The antibacterial properties were observed against pathogenic bacterial strains, viz., Escherichia coli, Bacillus subtilis, while the antifungal activity was done using fungal strains, viz., Fusarium oxysporum and Aspergillus niger. The disc diffusion method, as shown in Figures 8 and 9, was used for both activities. Thus, the findings indicate that the bacterial pathogen Escherichia coli was detected to have a maximum zone of inhibition in comparison to Bacillus subtilis (Table 1). In terms of antifungal activity, Aspergillus niger had the greater zone of inhibition when compared to Fusarium oxysporum (Table 2). a. Escherichia coli b. Bacillus subtilis Figure 8.Figure 8.Figure 8.Figure 8. Antibacterial activity of silver nanoparticles using Delonix regia shoot extract Sharma B and Alam A (2023). Not Sci Biol 15(1):11199 8 a. Fusarium oxysporum b. Aspergillus niger Figure 9Figure 9Figure 9Figure 9. Antifungal activity of shoot extract of Delonix regia Table 1. Table 1. Table 1. Table 1. Antibacterial activity of synthesized silver nanoparticles (AgNPs) S. No. Bacterial Species Zone of inhibition (mm) AgNPs (PE) Antibiotics (Chloramphenicol) Control (AgNO3) 1. Escherichia coli 13±0.21b 22±0.35c 12±0.08a 2. Bacillus subtilis 12 ±0.10b 21±0.29c 10±0.09a Table 2Table 2Table 2Table 2. Antifungal activity of synthesized AgNPs S. No. Fungal Species Zone of inhibition(mm) AgNPs (NP) Antibiotics (Fluconazole) Control (AgNO3) 1. Aspergillus niger 14±0.25b 25±0.46c 12± 0.09a 2. Fusarium oxysporum 10 ±0.08b 20± 0.32c 11 ±0.08a Statistical analysis The data are shown as averages of three replicates (n = 3). All obtained data were evaluated by IBM SPSS Statistics 20 software. Three-way interactions were executed between the chosen variables. For each output variable, multiple-comparison A Tukey's p<0.05 post hoc test was performed to compare the variance of the data. All data are presented as means standard deviation. Sharma B and Alam A (2023). Not Sci Biol 15(1):11199 9 DiscussionDiscussionDiscussionDiscussion Scientists are very interested in nanoparticles' significant antimicrobial impact because of the increasing microbial resistance to antibiotics and the development of antimicrobial resistant strains. One of the most encouraging approaches for avoiding microbial resistance is the use of nanoparticles, as they have the capability to fight drug resistance via multiple approaches. Silver nanoparticles (AgNPs) are highly useful in this context as they are effective antibacterial entities due to their enhanced reactivity, resulting from their high surface-to- volume ratio (Almatroudi, 2020). Hence, biosynthesized AgNPs emerge as one of the most viable alternative methods for the curing of microbial infections. In present study, we have investigated the UV-visible absorption spectra found near the wavelength of 425 nm. The particle size indicated its diameter, i.e., 129.6 nm, and the zeta potential of synthesized silver nanoparticles was investigated to be -18mv. The X-ray diffraction of synthesized silver nanoparticles revealed the intense peak at 38.50°, 39.94°, 44.36°, 64.42° and 77.44° and the average size of crystallized AgNPs was 35 nm. SEM study of silver nanoparticles disclosed that they are rectangular in shape and their size ranges from 40-60 nm. The Fourier Transform-Infrared spectrum of D. regia shoot extract revealed different peaks at 3363, 2920,1630 and 1472 cm-1 which indicates the presence of bonded OH-group, alkanes, carbonyl and amide bands. The antimicrobial activity showed the maximum zone of inhibition found in the bacterial activity, viz., E. coli and in fungal activity, the maximum zone of inhibition was found in A. niger. The aqueous shoot extract of D. regia was known to be an effective reducing and capping agent promoting efficient reduction of AgNPs and production of crystalline, spherical and stable at much lower concentration of plant extracts. Similar observations were noticed by other workers (Abu-Dief et al., 2021; Bala et al., 2017; Anitha and Sakthivel, 2016). ConclusionsConclusionsConclusionsConclusions The synthesis of silver nanoparticles has been found to be environmentally friendly and cost-effective, with numerous applications in fields such as biosensors, cosmetics, preservatives, antimicrobials, and so on. The aqueous shoot extract of Delonix regia was known to be an effective bio reducing and capping agent, promoting efficient reduction of silver nanoparticles and the production of crystalline, spherical, and stable plant extracts and silver salts at much lower concentrations. Various techniques, such as XRD, FTIR, and SEM, are used to characterize the biogenic synthesis of AgNPs. According to the findings of this study, plant-mediated synthesis of biogenic AgNPs has a variety of antimicrobial properties that are useful in pharmaceuticals and medical applications. Authors’ ContributionsAuthors’ ContributionsAuthors’ ContributionsAuthors’ Contributions Both authors came up with the idea and designed the piece. Sharma B carried out the experiment under Alam A's direct supervision. Sharma B performed all statistical analysis and drafted the manuscript. Both authors read and approved the final manuscript. Ethical approvalEthical approvalEthical approvalEthical approval (for researches involving animals or humans) Not applicable. Sharma B and Alam A (2023). Not Sci Biol 15(1):11199 10 AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements The authors would like to thank Professor Ina Shastri, Vice-Chancellor, Banasthali Vidyapith, Rajasthan, for her support and encouragement. We would also like to thank DST for providing networking assistance through the FIST programme at the Department of Bioscience and Biotechnology, Banasthali Vidyapith, as well as the DBT-funded Bioinformatics Center. Conflict of InterestsConflict of InterestsConflict of InterestsConflict of Interests The authors declare that there are no conflicts of interest related to this article. 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