Edith Egbunu

Advances in Bioscience and Bioengineering 2021; 9(4): 124-129 http://www.sciencepublishinggroup.com/j/abb doi:-/j.abb- ISSN:- (Print); ISSN:- (Online) Green Synthesis of Silver Nanoparticles Using Euphorbia hirta Leaf Extract and the Determination of Their Antimicrobial Activity Egbunu Iganya Edith1, *, Philip Felix Uzor1, 2 1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Madonna University, Elele, Nigeria 2 Department of Pharmaceutical and Medicinal Chemistry, University of Nigeria, Nsukka, Nigeria Email address: * Corresponding author To cite this article: Egbunu Iganya Edith, Philip Felix Uzor. Green Synthesis of Silver Nanoparticles Using Euphorbia hirta Leaf Extract and the Determination of Their Antimicrobial Activity. Advances in Bioscience and Bioengineering. Vol. 9, No. 4, 2021, pp. 124-129. doi:-/j.abb- Received: November 25, 2021; Accepted: December 14, 2021; Published: December 24, 2021 Abstract: Nanotechnology is a fast-growing field of science. Nanoparticles get much attention due to their unique physicochemical, optical and thermal activities. Silver nanoparticles have been used in experiments to treat infectious diseases. The goal of the research was to make silver nanoparticles using Euphorbia hirta extract, physically characterize the nanoparticles obtained and, to evaluate silver nanoparticles' antibacterial properties. The leaf extract of E. hirta (asthma weed) was used for the reduction of 1 mM silver nitrate (AgNO3) solution to silver nanoparticles (SNPs). SNPs were made by combining 50 mL of aqueous plant extract with 250 mL of AgNO3 solution to make SNPs. The mixture was monitored for two hours. The synthesized SNPs were characterized by UV-Vis, FTIR spectroscopy and particle size. The antimicrobial activity of the SNPs was tested against Escherichia. coli, Pseudomonas. aeruginosa, Salmonella. typhi, Bacillus. subtilis and Candida. albicans. The reaction medium's hue shifted from yellow to brown. The results of the UV-vis analysis of the particles showed that at 430 nm the particles had the maximum absorption (λ max) within 2 hours. The FTIR identified carboxylic acid and other functional groups. The polydispersity index (PDI) and Z-average particle size were found to be 0.426 and 274 nm, respectively. The results of the antimicrobial studies showed sufficient growth inhibition of the bacteria by the SNPs the minimum inhibitory concentration (MIC) ranging from 7 µg-10 µg. It was concluded that SNPs were synthesized using E. hirta leaf extract. The synthesized SNPs possess good activity against pathogenic microorganisms. Keywords: Nanoparticles, Synthesis, Extraction, Antimicrobial, Nanotechnology 1. Introduction The plant Euphorbia hirta originates from the family Euphorbiaceae; it is commonly known as asthma herb [1]. The greenish or reddish leaves (5 cm long) have oppositely arranged lanceolate clusters of flowers-like appearance [2]. The stem and leaves produce [3] white or milky juice when cut [4] The leaves are used to treat cough, asthma, worms, and vomiting. It has deworming properties, and it is also eaten as vegetables. The white latex is used as eye drops to cure conjunctivitis. Paste of leaf is applied externally on the place of scorpion bite. Euphorbia hirta. latex is rubbed on swellings, piles, and boils, and the root decoction is applied for snake bites, sores, wounds and is advantageous for nursing mothers with insufficient milk. The whole plant can be stipulated as an antidote; it is believed to be hemostatic, sedative, and soporific [5]. The manufacture of particles with at least one fraction in the range of 1–100 nm, resulting in a high surface-to-volume ratio, is known as nanotechnology [6]. Despite the diversity of metals found in nature, only a small percentage of them, such as gold, silver, palladium, and platinum, are widely synthesized in nanostructured form [7]. Silver nanoparticles have received a lot of attention since they can be utilized in a variety of applications, including pharmaceutics, agriculture, water detoxification, air filtration, textile industries, and as an oxidation catalyst [8]. Furthermore, one of their most important qualities is their antibacterial action against a wide spectrum of microorganisms without causing harm in animal cells. [9]. 125 Egbunu Iganya Edith and Philip Felix Uzor: Green Synthesis of Silver Nanoparticles Using Euphorbia hirta Leaf Extract and the Determination of Their Antimicrobial Activity Diverse parts of plants have been used to synthesize silver nanoparticles (SNPs) including [10], barks [11], seeds [12], etc. Different research papers have reported the antimicrobial activity of SNPs. The mechanism of antibacterial activity of SNPs is a source of debate that is currently poorly understood. There are, nevertheless, several assumptions and theories [13]. The goal of the study was to see how antimicrobial silver nanoparticles made from Euphorbia hirta extract performed. 2. Methodology prepared and autoclaved and poured into 13 agar plates; the different concentrations of the SNPs two standard drugs and the solvent (control) were added to the 13 different plates, one sample per plate. Then, the plates were allowed to solidify; Each plate was marked to divide into the different microorganisms; the different microorganisms were indicated by 1, 2, 3, 4, 5, 6 as six organisms were used (S. typhi, E. coli, B. subtilis, S. aureus, C. albicans, and A. niger). Each microorganism was streaked on the surface of each division of the agar plate, one microorganism per division. The plates were then incubated for 24 hours. 2.1. Preparation of Plant Extract 3. Results The leaves of the E. hirta plant were obtained from Heipang in Barkin Ladi LGA, Plateau State, Nigerian in October 2020. Following that, 20 g of powdered material was placed in 400 ml of distilled water, and the mixture was heated at 100°C for 30 minutes. Whatman filter paper no. 1 was used to filter the extract after it had been cooled. 3.1. Visual Observation and UV-visible Spectroscopy The reaction changed the color of the reaction mixture from yellowish-brown to dark brown, as shown in Figure 1, indicating that the Ag+ ion was reduced. 2.2. Green Synthesis of Silver Nanoparticles Silver nitrate (0.1698 g) was weighed accurately with an analytical weighing balance and dissolved with distilled water in a 50 ml beaker; it was then transferred to a 1 L volumetric flask where the volume of the solution was made up to the 1 L mark of the volumetric flask to make 1mM solution. The SNPs solution provided the silver ion for the reaction. The plant extract (50 ml) was mixed with 250 ml of AgNO3 solution 1 mM. The reaction was incubated at a temperature of 27°C in the dark to avoid photochemical activation of AgNO3. Colour change to dark brown was taken as an indication of synthesis of SNPs. The obtained solution was centrifuged for 30 minutes at 5000 rpm. To remove silver ions and seed extract residue, the pellet containing silver nanoparticles was washed 3–4 times with distilled water. Figure 1. Change in color after reducing Ag⁺ to silver nanoparticle by using Euphorbia hirta leaf extract. Keys:(A) at 0 minutes, (B) at 30 minutes, (C) at 1 hour. The ultraviolet-visible spectroscopy graph at 0 hour Figure 2 shows the initial onset of reaction; it can be seen that for the initial onset, a valley was observed before a sudden peak in the reaction at about 430 nm this is also reflected in Figure 8 which shows the combined graph of the reaction from 0 to 2.5 h. 2.3. Ultraviolet-visible Spectroscopy (UV-Vis) The extracted SNPs were analyzed by scanning under a wavelength ranging from 300-800 nm using the UV-VIS spectrophotometer (JENWAY, 6705) at 30 minutes for 2 hours. 2.4. Particle Size Determination Dynamic light scattering was used to figure out the particle size (D. L. S.). In addition, the polydispersity index and Z-average were recorded. The instrument used for the analysis was the (Malvern Nano ZS7.01). 2.5. Fourier Transformed Infrared Spectroscopy (FTIR) Figure 2. UV-Vis Spectroscopy at 0 hour. In Figures 3-7, it can be seen that absorbance increased with time (0.5-2.5h) almost the same wavelength as in Figure 2; this indicated an increase in the amount of the SNPs synthesized with an increase in time. The FTIR analysis of the SNPs was done using the FTIR-8400 S spectrophotometer system. 2.6. Antimicrobial Analysis Agar diffusion method was adopted for the antimicrobial assay. The silver nanoparticles extracted were diluted at ten (10) different concentrations (1, 2, 3…10 µg/ml). The control drugs used were (fluconazole and ciprofloxacin). The agar was Figure 3. UV-Vis spectroscopy at 0.5 hour. Advances in Bioscience and Bioengineering 2021; 9(4): 124-129 126 3.2. Particle Size Analysis The Z-average of the silver nanoparticles was 274.6 nm (diameter), and the polydispersity index was 0.426, according to dynamic light scattering, commonly known as photon correlation spectroscopy analysis. Figure 9. Figure 4. UV-Vis spectroscopy at 1 hour. Figure 5. UV-Vis Spectroscopy at 1.5 hour. Figure 9. Particle size distribution by intensity for synthesized silver nanoparticles (SNPs). Table 1. Particle size distribution by intensity. Peak 1 Peak 2 Peak 3 Z-average (d.nm) PDI Particle size range Figure 6. UV-Vis Spectroscopy at 2 hours. Figure 7. UV-Vis spectroscopy at 2.5 hours. Size (d.nm- <100nm Intensity (%- From the analysis carried out, it can be noted that the average particle size of the E. hirta silver nanoparticle extract was 274.6 d.nm (Z-average). From Table 1 above, it can be observed that the intensity of peak 1 was 68%, and in the graph of Figure 9, the highest intensity of the particles was within a range of 90-120 nm (diameter), the aggregation of the particles over time resulted in a subsequent increase in particle size. The numerical value of PDI for the extract was 0.426, which is within the range for nanoparticles indicating that even though the particles are not evenly distributed, they have some form of uniformity in distribution. 3.3. Results of FTIR The results of FTIR analysis are presented in Table 2. The study was conducted to discover the various functional groups found in SNPs and their role in the stability and production of silver nanoparticles. Several peaks indicating the complex nature of the biological material. The stretching vibrations of the C-O and OH link carboxylic acid, alcohol, 2345.52 nitrile, N-O nitro compound, 3441.12 N-H amine group, Si-OH silanol are ascribed to the bands occurring at 1103.32, 3178.79, 3294.53, 3510.56, 3626.29, and 3772.89 cm-1, respectively. Figure 8. UV-vis absorbance characteristics displayed by AgNPs of E. hirta leaf extract at different time interval. 127 Egbunu Iganya Edith and Philip Felix Uzor: Green Synthesis of Silver Nanoparticles Using Euphorbia hirta Leaf Extract and the Determination of Their Antimicrobial Activity Table 2. FTIR analysis of silver nanoparticles obtained using E. hirta leaf extract. Peak number-. Absorption frequency (cm- Area- Intensity- 3.4. Antimicrobial Assay The MIC of antimicrobial drugs against various bacteria was determined using the Agar dilution method. S. typhi had a MIC of 10 g/ml, E. coli 9 g/ml, B. subtilis 7 g/ml, S. aureus 9 g/ml, and C. albicans 9 g/ml after 24 hours of incubation. 4. Discussion SNPs have been synthesized with different varieties of plants such as Azadirachta indica, Annona muricata, Origanum vulgare, e. t. c. The creation of silver nanoparticles began with the addition of Euphorbia hirta leaf extract to 1 mM silver nitrate (AgNO3). The gradual shift in color of the mixture from yellowish to reddish-brown, as depicted in Figure 1, indicated the production of SNPs. Surface Plasmon (i.e., quantum of plasma oscillation) vibration causes the color change, which is an optical feature specific to noble metals. [14]. To get a decent outcome, several parameters had to be tuned, including the concentration of AgNO3, E. hirta leaf extract, and time, all of which have been identified as factors impacting SNP production. It was also observed in other plant extracts that heating of the reaction mixture also increased the yield of silver nanoparticles. UV-Vis spectroscopy was used to further examine the sample at various time intervals. The samples had valleys within 300-400 nm wavelength and peaks at 400-500 nm wavelength; the maximum wavelength from the combined graph see Figure 8 above was 430 nm which is characteristic of SNPs [15]. The curve in Figure 8 showed that with an increase in incubation time there was an increase in absorbance which was similar to previous reports. [13], The reaction lasted for 2.5 hours, the same time interval was seen in earlier study [16]. A range of factors could have affected the UV-Vis absorption characteristics of the extract; these could include; solvent, concentration, pH, temperature. By modifying the position (ℷ max) and intensity (ℷ max) of the chromophore's absorption peaks in the compound, Possible bond C-H C-H C-O C=C C=C N-O R─N═C═S C-H C≡N C-H C-H O-H O-H N-H O-H O-H O-H Si-OH Si-OH Possible functional group Alkene Alkene Alcohol, carboxylic acid, ether Alkene Alkene Nitro compound Isothiocyanates Alkene Nitrile Alkane Alkene Carboxylic group Carboxylic group, H- bonded Amines Carboxylic acid Alcohol Alcohol, carboxylic acid, free hydroxyl group Silanol Silanol uncontrolled alterations in these factors can cause inaccuracies. These parameters were adequately controlled in order to obtain a meaningful and quantitative result [17]. A broad peak at a higher wavelength usually implies an increase in particle size, whereas a thin line at a shorter wavelength usually suggests a decrease in particle size. Other studies have shown that raising the percentage of plant extract can greatly influence the size and size distribution of nanoparticles [18]. A broad peak at a longer wavelength usually indicates an increase in particle size, whereas a thin line at a shorter wavelength usually indicates a decrease. Other research has found that increasing the proportion of plant extract in nanoparticles has a significant impact on their size and size dispersion [19]. The presence of different primary functional groups in the SNPs and their likely participation in the synthesis and stability of SNPs was detected using Fourier transform infrared spectroscopy (FTIR). The functional groups observed are comparable to those seen in prior investigations [20]. The existence of O–H stretch and hydrogen-bonded groups in alcohol, phenolic, or water molecules in the extract can be seen in the broad stretch at 3294 cm-1 [21]. The coordination of SNPs with – OH and C=O groups may be responsible for the stability and capping agent of synthesized SNPs. It's also possible that the reduction process is caused by the presence of phenolic and flavonoid group molecules; the phenol present has disinfectant capabilities [22]. Antioxidant, anti-allergic, anti-cancer, anti-inflammatory, and antiviral effects are all found in flavonoids. For example, the flavonoid quercetin has been shown to help with asthma, hay fever, and sinusitis symptoms [23]. Antibacterial testing on the SNPs revealed impressive antimicrobial activity, however it was lower than that of the control antibiotics (gentamicin and ofloxacin) utilized in Table 2 above. Biogenic SNPs have previously been shown to be more effective antibacterial agents than chemically produced SNPs [24]. In the results of the P. aeruginosa and E. coli were found to have the highest sensitivity to the synthesized SNPs, while C. albicans was more resistant to the synthesized SNPs Advances in Bioscience and Bioengineering 2021; 9(4): 124-129 [14]. The minimum inhibitory concentration (MIC) of SNPs against S. typhi, E. coli, B. subtilis, S. aureus, C. albicans, and A. niger was determined after the antibacterial activity of produced AgNPs was confirmed using a disc diffusion experiment. The MIC of antimicrobial drugs against diverse bacteria was determined using the agar dilution method. After a 24-hour incubation period, the MIC was noted, S. typhi had a MIC of 10µg/ml, E. coli 9µg/ml, B. subtilis 7µg/ml, S. aureus 9µg/ml and C. albicans 9µg/ml. 5. Conclusion Based on the study carried out above E. hirta leaf extract was shown to be an efficient reducing agent for the synthesis of SNPs; the SNPs were made utilizing E. hirta leaf extract.,. The synthesized SNPs showed remarkable stability. The nanoparticles showed profound antimicrobial activity that could be used to develop antimicrobial drugs. The procedure for producing silver nanoparticles was found to be easy, quick, eco-friendly, and non-toxic. 6. Recommendation Further research can be carried out on the silver nanoparticles to make them useable in the formulation of creams or ointments for the treatment of infections caused by sensitive organisms. Acknowledgements We thank the Madonna University Pharmaceutical Chemistry and the Pharmaceutical Microbiology laboratory staff for providing us with the necessary apparatus and equipment and their support during the research. References [1] Soforowa et al, (1993) "Recent Trends in Research into African Medicinal Plants," Journal of Ethnopharmacolgy, pp. 28 (1-2): 197-208. [2] Gupta et al, (2019) “Investigattion of the Antimicrobial Activity of Euphorbia hirta leaves,” International Jorunal of Life Science and Pharma Research, pp. 9 (3): 32-37. [3] Ammar Altemimi, Naoufal Lakhssassi, Azam Baharlouei, Dennis G. Watson and David A. Lightfoot, (2017) "Phytochemicals: Extraction, Isolation, and Identification of Bioactive Compoundsfrom Plant Extracts," plants, p. 6 (4): 42. [4] Lind E. M. and A. C. Tallantire A. C. (1971), “Some Common Flowering Plants of Uganda,” oxford university press, Nairobi. [5] Linfang Huang, Shilin Chen, and Meihua Yang, (2012) "Euphorbia hirta (Feiyangcao): A Review on its Ethnopharmacology, Phytochemistry and Pharmacology," Journal of Medicinal Plants Research, pp-. [6] Narayanan, K. S. N. (2010) "Biological Synthesis of Metal Nanoparticles by Microbes.," Advance Colloid Interface Science, pp. 156 (1-2): 1-13. 128 [7] Yang Y., Jin P., Ravichandran N., Ying H., Yu C. Wang K (2017)., "New epigallocatechin gallate (EGCG) nanocomplexes co-assembled with 3-mercapto 1-hexanol and β-lactoglobulin for improvement of antitumor activity," Journal of Biomedical Nanotechnology, pp. 13 (7): 805-814. [8] Arvizo, (2012) "Intrinsic Therapeutic Applications of Noble Metal Nanoparticles: Past, Present and Future," Chemistry Society Reviews, pp. 41 (7):-. [9] Elshawy O. E, (2016) "Preparation, Charac terization and Invitro Evaluation of the Antitumor Activity of the Biologically Synthesized Silver Nanoparticles," Advancement of Nanoparticles, pp. 5 (02): 149-166. [10] Suman T, (2013) "Biosynthesis, characterization and cytotoxic effect of plant mediated silver nanoparticles using Morinda citrifolia root extract.," Colloids Surfaces B: Biointerface, pp. 106: 74-78. [11] Sathishkumar M, et al., (2009) "Cinnamon zeylanicum bark extract and powder mediated green synthesis of nano-crystalline silver particles and its bactericidal activity," Colloids Surfaces B: Biointerfaces, p. 73 (2): 332. [12] Bar H, et al, (2009) "Green synthesis of silver nanoparticles using seed extract of Jatropha," Colloids Surf A Physicochemical and Engineering Aspects, pp. 348 (1-3): 212-216. [13] Akhil Rautela, (2019)"Green synthesis of silver nanoparticles from Tectona grandis seeds extract: characterization and mechanism of antimicrobial action on different microorganisms," Journal of Analytical Science and technology, pp. 10 (1): 1-10. [14] Ibrahim HM, (2015) "Green Synthesis and characterization of Sliver nanoparticles using banana peel extract and their antimicrobial activity against representative microorganisms," Journal of radiation Research and Applied Science, pp. 8 (3): 265-275. [15] Gaurav Bahuguna, Amit Kumar, Neeraj K Mishra, Chitresh Kumar, Aseema Bahlwal, Pratibha Chaudhary and Rajeev Singh, (2016) "Green synthesis and characterization of silver nanoparticles using aqueous petal extract of the medicinal plant Combretum indicum," Material Research Express, p. 3 (7): 075003. [16] Mohammed Rafi Shaik 1 ID, Mujeeb Khan, Mufsir Kuniyil ID, Abdulrahman Al-Warthan Hamad Z. Alkhathlan, (2018) "Plant-Extract-Assisted Green Synthesis of Silver Nanoparticles Using Origanum vulgare L. Extract and Their Microbicidal Activities," Sustainability, p. 10 (4): 913,. [17] Smitha, S.; Nissamudeen, K.; Philip, D.; Gopchandran, K, (2008)"Studies on surface plasmon resonance and photoluminescence of silver nanoparticles," Spectrochim. Acta Part A Molecular and Biomolecular Spectroscopy, pp. 71 (7): 186-190. [18] Prathna, T.; Chandrasekaran, N.; Raichur, A. M.; Mukherjee, A. (2011), "A. Biomimetic synthesis of silver nanoparticles by Citrus limon (lemon) aqueous extract and theoretical prediction of particle size.," Colloids Surf. B Biointerfaces, pp. 82 (1): 152-159. [19] Souza T. G. F., Ciminelli V. S. T., N. D. S. Mohallem, (2016) "A comparison of TEM and DLS methods to characterize size," Journal of Physics: Conference Series, pp. 733 (1):-. 129 Egbunu Iganya Edith and Philip Felix Uzor: Green Synthesis of Silver Nanoparticles Using Euphorbia hirta Leaf Extract and the Determination of Their Antimicrobial Activity [20] Le Thi Bach, Bui Thi Buu Hue, Nguyen Thi Thu Tram, Do Nguyen Anh Thu, and Le Tien Dung, (2020) "Chemical constituents from n-hexane and ethyl acetate extracts," Conference Series: Materials Science and Engineering, pp. 736 (2): 022-083, 2020. [21] Singh P, Kim Y J, Zhang D., (2016) "Biological synthesis of nanoparticles from plants and microorganisms," Trends Biotechnology, pp. 34 (7): 588-599. [22] Rastogi L. and Arunchalam J., (2011) "Sunlight based irradiation strategy for rapid green synthesis of highly stable silver nanoparticles using aqueous garlic," Material Chemistry and Physics, pp. 129 (1-2): 558-563. [23] Jhoanna Robinson, (2017) "Flavonoids sources, health benefits and uses," Naturalpedia. [24] Bawskar M, Deshmukh S, Bansod S, Gade A, Rai M, (2015) "Comparative analysis of biosynthesized and chemosynthesized," IET Nanobiotechnology, pp. 9 (3): 107-113.