Sundus H. Ahmed, Rasha S. Hameed, Alyaa M. Yousif, Zena H. Gazar
Mustansiriyah University, College of Science, Biology Department
In our current study, green nanoparticles were prepared using lettuce Lactuca sativa without using toxic chemicals. Visually, the formation of silver nanoparticles was confirmed by observing changes in color from pale yellow to dark brown. The peak absorption of the UV spectrophotometer was observed at 360 nm. Nanoparticles were characterized by SEM and FTIR analysis. Antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris, a Bacillus subtls and Klebsiella pneumoneae, Candida spp and phytopathogenic Aspergillus niger; the prepared nanoparticles showed a high anti-inflammatory effect compared to the standard drug Profein. The results suggest that silver nanoparticles may have an important advantage compared to traditional antibiotics, and it was concluded the the nano particle accumulate the active component of Lactuca sativa may be for this it gives the highest scavenging activity.
Numerous studies have shown a correlation between the consumption of fresh fruits and vegetables and their health benefits. Epidemiological studies have further demonstrated the relationship between dietary habits and disease risk and established that food has a direct impact on health. Lettuce, Lactuca sativa, is an important dietary leafy vegetable that is primarily consumed fresh or in salad mixes due to its perception as being amongst healthier foods[1,2]. A number of lettuce varieties have been investigated recently and reported to contain phenolic compounds with antioxidant activities[3,4].
In recent years, demand for high quality lettuce with minimal or no pesticide residues, has also risen sharply to serve local consumption and for export. Therefore, better agricultural practices for disease management with consideration on maintaining a more sustainable and healthier crop eco-system should be taken. Developing alternative strategies to improve plant disease resistance and control of pathogens would be promoted[5-7].
In our current study, green nanoparticles were prepared using lettuce Lactuca sativa without using toxic chemicals, The results suggest that silver nanoparticles may have an important advantage compared to traditional antibiotics.
MATERIALS AND METHODS:
Plant material and synthesis of silver nanoparticle:
The Lactuca sativa leaves were ground to a fine powder. Silver nitrate1 mM was added to the plant extract to make up a final solution of 200ml and centrifuged at 10,000 rpm for 25 min. The supernatants were heated at 80ºC. A change in the color of the solution was observed during heating of process within 10-15 minutes. The color changes indicate the formation of silver nanoparticles (SNPs). The reduction of pure Ag2+ ions were monitored by measuring the UV-Vis spectrum of the reduction media at 5 hours after diluting a small aliquot of the sample in distilled water by using 118 UV-Vis Spectrophotometer.
Culture of, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoneae species of bacteria and Aspergillus niger and Candida spp.
The antibacterial activities of SNPs were carried out by disc diffusion method 14. Nutrient agar medium plates were prepared, sterilized and solidified. After solidification bacterial cultures were swabbed on these plates. The sterile discs were dipped in silver nanoparticles solution (10mg/ml) and placed in the nutrient agar plate and kept for incubation at 370C for 24 hours. Zones of inhibition for control, SNPs and silver nitrate were measured. The experiments were repeated thrice and mean values of zone diameter were presented .
Potato dextrose agar plates were prepared, sterilized and solidified, after solidification fungal cultures were swabbed on these plates with 40cell/ml. The sterile discs were dipped in silver nanoparticles solution (10mg/ml) and placed in the agar plate and kept for incubation for 7 days. After 7 days zone of inhibition was measured.
Synthesis of silver Nanoparticle:
About 3ml of Lutica extract was added to 20ml of of AgNO3 (0.5Mm). The mixture was boiled at 80°C for 20 minutes, while heating the colour of solution was changed from pale brown to dark brown. The reduction of Ag+ ions to Ag0 was monitored by measuring the UV-vis spectrum of various concentration of reaction mixture (silver nitrate solution leave extract).
UV-Vis Spectra analysis:
Vis spectral analysis was done by using UV-Vis spectrophotometer at the range of 300-700 nm and observed the absorption peaks at 300nm regions, which are identical to the characteristics UV-visible spectrum of metallic silver and it was recorded.
SEM analysis of silver nanoparticles:
Thin films of the sample were prepared on a carbon coated copper grid by just dropping a very small amount of the sample on the grid, extra solution was removed using a blotting paper and then the film on the SEM grid were allowed to dry for analysis.
FTIR analysis of silver nanoparticles:
For FTIR measurements, the synthesized silver nanoparticles solution was centrifuged at 10000 rpm for 30 minutes. The pellet was washed thrice with 5ml of deionized water to get rid of the free proteins or enzymes that are not capping the silver nanoparticles. The pellet was dried by using vacuum drier. This was analyzed by FTIR.
RESULTS AND DISCUSSION:
Synthesis of silver nano particles:
The reduction of silver ions into silver particles during exposure to the plant extract is followed by color change from pale yallow to brown color. It is well known that silver nanoparticles exhibit yellowish brown color in aqueous solution due to excitation of surface plasmon vibrations in silver nanoparticles. As the plant extract was mixed in the aqueous solution of the silver ion complex, it started to change the color due to reduction of silver ion, which may be the indication of formation silver nanoparticles there by leading to the formation of silver hydrosol (9) Fig 1. The UV-Vis spectrum of colloidal solutions of SNPs synthesized from Lactuca sativa have an intense peak was observed in the UV-spectrophotometer at 300nm (Fig 2).
Figure 1: Absorbance of sample vs wave length.
Figure 2: Biosynthesis of nanoparticle
The SEM image of silver nanoparticles were synthesized from Lactuca sativa extract were assembled on to the surface due to the interactions such as hydrogen bond and electrostatic interactions between the bio-organic capping molecules bound to the Ag nanoparticles. It was shown that relatively spherical and uniform silver nanoparticles were formed. The nanoparticles were not in direct contact even within the aggregates, indicating stabilization of the nanoparticles by a capping agent[10-12].
The scanning electron microscopic (SEM) image shown high density Ag nanoparticles synthesized by Lactuca sativa plant extracts further confirmed the presence of Ag nanoparticles (Fig 3). It was shown that relatively spherical and uniform Ag nanoparticles were formed with diameter of 15 to 25.6 nm.
Figure 3: SEM analysis, it was shown that relatively spherical and uniform silver nanoparticles were formed.
Fig. 4. shows the antimicrobial activity of synthesized Ag nanoparticles against five different bacteria and fungi such as Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris and Klebsiella pneumoneae, Candida spp and phytopathogenic Aspergillus niger. As it showed a clear inhibition zone, the synthesized Ag nanoparticles were highly effective in their activity against Pseudomonas aeruginosa> Escherichia coli, > and Klebsiella pneumoneae, Candida spp. and phytopathogenic Aspergillus niger and. The silver nanoparticles synthesized via green route are highly toxic towards fungal species also when compared to bacterial species. The ionic silver strongly interacts with thiol group of vital enzymes and inactivate the enzyme activity [13, 14]. Experimental evidence indicates that DNA loses its replication ability once the bacteria have been treated with silver ions. our findings of suggested that the inhibition of oxidation based biological process by penetration of metallic nano sized particles across the microsomal membrane.
Figure 4: Inhibition activity of Lactuca sativa activity extract, and silver nano particle 3Mm and 2m(Ps)-Pseudomonas aeruginosa, (Ec) Escherichia coli, (Kp)- Klebsiella pneumoneae (An) Aspergillus niger (Af), (C) Candida spp.
Fig. 5 shows the amount of each sample analyzed needed for 50% inhibition (IC50). IC50 of Lactuca sativa the highest radical scavenging activity was observed by nanoparticle significantly higher than extract (P>0.05) the scavenging activity of nanoparticle exhibited more than 87% scavenging followed in compare with extract about 55% may be the nano particle accumulate the active component of Lactuca sativa may be for this it gives the highest scavenging activity.
Figure 5: Antioxidant activity of Lactuca sativa extract and synthesis Nanoparticle.
FTIR analysis FTIR spectrum of Ag nanoparticles synthesized from Lactuca sativa extract were shown in Fig(6). FTIR measurements were carried out to identify the possible biomolecules responsible for capping and efficient stabilization of the metal nanoparticles synthesized by leave extract. The Peaks near 3440 cm-1, 2924cm-1 and 2854-1 assigned to OH stretching and aldehydic C-H stretching respectively. The weaker band at 1629 cm-1 corresponds to amide I arising due to carbonyl stretch in proteins. The peak at 1041 cm-1 corresponds to C-N stretching vibration of the amine. The peak near 1741 cm-1 corresponds to C=C stretching (non conjugated). The peak near 833 cm-1 assigned to –C=CH2. the peak near 677cm-1 and 651.96 cm-1 assigned to CH out of plane bending vibrations are substituted ethylene systems -CH=CH(cis) .
Figure 6: FTIR spectra of silver nanoparticles synthesized by of Lactuca sativa extract
The researchers would like to acknowledge Mustansiriyah University for supporting the researchers of this work in providing raw materials as well as using the apparatuses and labs to achieve this work.
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Received on 21.05.2019 Modified on 10.06.2019
Accepted on 01.07.2019 © RJPT All right reserved