{"id":31255,"date":"2020-03-28T10:16:31","date_gmt":"2020-03-28T10:16:31","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=31255"},"modified":"2020-04-22T12:32:39","modified_gmt":"2020-04-22T12:32:39","slug":"studies-on-antioxidant-and-antimicrobial-potential-of-biogenic-silver-nanoparticles-synthesized-using-nothapodytes-foetida-leaf-extract-wight-sleumer","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol13no1\/studies-on-antioxidant-and-antimicrobial-potential-of-biogenic-silver-nanoparticles-synthesized-using-nothapodytes-foetida-leaf-extract-wight-sleumer\/","title":{"rendered":"Studies on Antioxidant and Antimicrobial Potential of Biogenic Silver Nanoparticles Synthesized using Nothapodytes Foetida Leaf Extract (Wight) Sleumer"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

In recent years biogenic nanomaterials have emerged with an immense scope in biomedicine as these nanostructures are proved to combat against deadly communicable and non-communicable diseases. Intensifying application of antibiotics in therapeutics endorsed multidrug resistance (MDR) in numerous microbial strains especially in bacterial pathogens. The abridged efficacy of drugs evidenced that the MDR bacteria could be a major warning to the human wellbeing.1 <\/sup>Therefore, to cope up with the threat of pathogenic executors, pioneered domain of nanotechnology has come up with an increasingly compelling and non-toxic treatment where biogenic nanomaterials are of enormous concern in the field of biomedical research because of their biological origin, mode of action and response. The biomaterials including phytochemicals with natural origin are gifted products of medicinal plants come forward to battle against deadly infectious microorganisms.2-3<\/sup> Various attempts have been made synthesize different metal nanoparticles using biomaterials and to explore their antioxidant and antimicrobial potential.4-5<\/sup> Now days, bio-synthesis of nanoparticles has become a keen interest of research for their advanced application in curative treatment to overcome the overall burden of deadly infectious diseases. Biogenic nanoparticles synthesized using extracts of medicinal plants are significantly illustrated for their biological efficacies including antimicrobial and antioxidant potential6-8<\/sup> but, still there remains paucity in the knowledge on application of biogenic silver nanoparticles synthesized using Nothapodytes foetida<\/em>.<\/p>\n

Nothapodytes foetida <\/em>(Wight) Sleumer is an important endangered medicinal plant with extensive antioxidant9<\/sup> and cytotoxic potential10<\/sup> with high therapeutic value because of abundance in active constituents including camptothecin and its derivatives predominantly used in clinical complications. Earlier, studies on biogenic silver nanopartiles synthesized using Nothapodytes<\/em> species, reported cytotoxicity potential.11-12<\/sup> But, the documentary evidences on antioxidant and antimicrobial potential of silver nanoparticles synthesized using any of the Nothapodytes<\/em> species remained unexplored till date. Therefore in this study we focused our attention at synthesizing silver nanoparticles using crude aqueous leaf extract of N. foetida<\/em> and subsequently explore for their biological properties including antioxidant and antimicrobial potential.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Biosynthesis and purification of silver nanoparticles<\/strong><\/p>\n

The silver nanoparticles were synthesized by adding 10 mL of aqueous plant extract of N. foetida<\/em> to 90 mL of 1 mili molar (mM) silver nitrate (AgNO3) solution. The preparation was then incubated at 800<\/sup>C in dark and monitored continuously for synthesis of nanoparticles with change in colour of reaction mixture. After formation of NFAgNPs, the mixture was centrifuged at 15000 rpm for 10 minutes and washed several times with sterile deionized distilled water (ddw) to remove unwanted traces of contaminants followed by redispersion of the pellet in sterile ddw for further characterizations.<\/p>\n

Visual Observation<\/strong><\/p>\n

The reduction of silver nitrate using aqueous plant extract was monitored for time periods (10, 30, 60, 120 and 180 minutes) and the appearance of dark brown color indicates the formation of silver nanoparticles.<\/p>\n

UV-Visible Spectral Analysis<\/strong><\/p>\n

Preliminary characterization of biosynthesized AgNPs was carried out using UV-Visible spectroscopy. The reduction of pure silver ions was monitored by measuring UV visible spectrum of the reaction mixture at a wavelength of 300-800 nm by sampling the aliquots withdrawn from reaction mixture at different time intervals. The measurements were recorded on UV\u2013visible Dual Beam Spectrophotometer (UV-1800 Shimadzu).<\/p>\n

Characterization of NFAgNPs <\/strong><\/p>\n

Characterization of biosynthesized NFAgNPs were carried out by Transmission Electron Microscopy (TEM) at Sophisticated Analytical Instrument Facility at Sophisticated Test and Instrumentation Centre, Cochin University, Kerala.<\/p>\n

Antioxidant activity of NFAgNPs<\/strong><\/p>\n

The antioxidant activity of NFAgNPs was characterized by DPPH & ABTS assay.<\/p>\n

DPPH Radical Scavenging Assay <\/strong><\/p>\n

In vitro<\/em> reaction of DPPH assay consisted of a mixture of freshly prepared 1 mL DPPH (0.1 mM) solution in methanol with different concentrations (5, 10, 25, 50, 75 and 100 \u03bcg\/mL of NFAgNPs in final volume of 0.1 mL in distilled water. The mixture was shaken vigorously and allowed to stand at room temperature for 30\u2009min in dark thereafter the absorbance was measured at 517 nm by using a UV-Visible 1800 spectrophotometer (Shimadzu). The percentage of DPPH scavenging activity was calculated using the formula: Percentage inhibition (%) = (A0-A1) \/ A0) \u00d7 100, where: A0 is the Absorbance of control and A1 Absorbance of test. The results were compared with Butylated hydroxy toluene (BHT) as standard<\/p>\n

ABTS Radical Scavenging Assay<\/strong><\/p>\n

ABTS radicals are produced by reacting ABTS (7 mM) and potassium persulfate (2.4 mM) and incubating the mixture at room temperature in the dark for 16 hr. Different concentrations of NFAgNPs in final volume of 0.1 mL were added and allowed to react with 1 mL of ABTS for 30 minutes and the absorbance was recorded at 734 nm after incubation, thereafter calculated percent inhibition of ABTS radicals using BHT as standard.<\/p>\n

Evaluation of Antimicrobial Properties of NFAgNPs<\/strong><\/p>\n

The antimicrobial activity of NFAgNPs was performed against pathogenic Gram-positive bacteria Staphylococcus aureus<\/em> (S. aureus<\/em>) (ATCC\u00ae 29213\u2122) and Gram-negative bacteria, Escherichia coli<\/em> (E. coli<\/em>) (ATCC\u00ae 25922\u2122), Klebsiella pneumoniae <\/em>(K. pneumoniae<\/em>) (ATCC\u00ae 700603\u2122) and Pseudomonas aeruginosa<\/em> (P. aeruhinosa<\/em>) (ATCC\u00ae 2617\u2122) by minimum inhibitory concentration (MIC) assay.<\/p>\n

Statistical Analysis<\/strong><\/p>\n

All experiments were done in four replicates and then values were expressed as mean \u00b1 standard error (SE) of four measurements. Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Student\u2019s t-test by SPSS 11 for windows.<\/p>\n

Results<\/strong><\/p>\n

Biosynthesis & Characterization of NFAgNPs<\/strong><\/p>\n

Aqueous leaf extract of N. foetida<\/em> was used for biosynthesis of silver nanoparticles without any other stabilizing agent. After adding faint yellow plant extract to clear solution of AgNO3 solution changed to dark brown color within 3 hours of incubation at 800<\/sup>C (Figure 1A and C) demonstrated bioreduction of silver ions to nanosilver which is accelereted by the N. foetida<\/em> leaf extract. The change in color of the AgNO3 solution after addition of aqueous leaf extract of N. foetida<\/em> confirms the formation of silver nanoparticles which may occurred because of phytochemicals present in N.<\/em> foetida<\/em> might be responsible for reduction and synthesis of AgNPs.<\/p>\n

The primary characterization of NFAgNPs synthesized using aqueous extract of N. foetida<\/em> was done by UV-visible spectroscopy. The time dependent extinction of surface plasma resonance in biosynthesized NFAgNP solutions was indicated in UV- visible spectra (Figure 1B & D) which exhibited a characteristic absorbance maxima at 440 nm for NFAgNPs at various time points which showed synthesis of nanoparticles initiated after 30 minutes of incubation and the intensity increases progressively without any shift of the peak and completed after 180 minutes of incubation. The morphology and shape of biosynthesized NFAgNPs were determined by TEM which shows that the biosynthesized NFAgNPs are spherical in shape and 20-50 nm size.<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 1: A.<\/strong> Biosynthesis of NFAgNPs, bottle containing N. foetida<\/em> plant extract, sample of 1mM AgNO3 and colloidal solution of NFAgNPs.B.\u00a0<\/strong>Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 2: TEM analysis of NFAgNPs<\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

In Vitro <\/em>DPPH Radical Scavenging Activity<\/strong><\/p>\n

The results of the DPPH and ABTS radical scavenging activity of NFAgNPs are shown in figure 3A & B. As shown in figure 3A, maximum percentage scavenging of DPPH radicals by NFAgNPs was 22.70 \u00b1 1.76, 36.40 \u00b1 1.73, 46.81 \u00b1 1.30, 65.17 \u00b11.90, 81.80 \u00b1 1.04 and 93.80 \u00b1 1.98 for 5, 10, 25, 50, 75 and 100 \u00b5g\/mL concentrations respectively.<\/p>\n

ABTS<\/strong> Radical Scavenging Activity <\/strong><\/p>\n

As shown in figure 3B, maximum percentage scavenging of ABTS for NFAgNPs was 84.59%, at 100 \u00b5g\/mL followed by 72.25%, 59.19%, 43.38%, 32.13% and 17.53% at decreasing concentrations of NFAgNPs i.e. 75, 50, 25, 10 and 5 \u00b5g\/mL respectively. The results showed that the NFAgNPs exhibited strong DPPH radical scavenging ability with 50% inhibitory concentration IC50<\/sub> 22.56 \u03bcg\/mL than the ABTS radical scavenging activity (IC50 value of 41.47\u03bcg\/mL).<\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 3: <\/strong>Representative histogram showing.<\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Antibacterial activity of NFAgNPs.<\/strong><\/p>\n

The antibacterial activity of the NFAgNPs was assayed in vitro<\/em> by minimum inhibition concentration method against four bacteria species, E. coli P. aeruginosa, K. pneumoniae<\/em> and S. aureus.<\/em> Different concentrations of NFAgNPs (0.1, 0.2, 0.4, 0.8 and 1.0 mg\/mL) were tested on Muller Hinton media along with amphicillin as positive control. The results of antibacterial activity of biogenic NFAgNPs were represented in Figure 4A-D. The MIC results indicated that higher concentrations of NFAgNPs (1.0 mg\/mL) showed greater sensitivity of E.coli<\/em> P. aeruginosa<\/em> strains and K. pneumonia<\/em>e whereas S. aereus<\/em> required higher concentrations of NFAgNPs to inhibit bacterial cell growth. When we tested antibacterial effect of NFAgNPs, we observed the highest percentage of bacterial growth inhibition in E.coli<\/em> (95.03 \u00b1 1.16), P. aeruginosa<\/em> strains (94.24 \u00b1 1.27) and K. pneumoniae<\/em> (91.90 \u00b1 2.69) when exposed to 1 mg\/mL concentration whereas 63.02 + 0.50 % growth inhibition occurred in S. aureus<\/em>.<\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 4:<\/strong> Representative histogram showing bacterial growth inhibition of.
\n<\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Discussion<\/strong><\/p>\n

Present work explains antioxidant and antibacterial potential of biogenic AgNPs synthesized using N. foetida<\/em> leaf extract. After addition of aqueous extract of N. foetida<\/em>, the color of silver nitrate solution turned to dark brown after 3 h of incubation indicating formation of silver nanoparticles. There was no change in color of the reaction observed up to 10 minutes after addition of plant extract to silver nitrate solution which started after 30 minutes of incubation in dark at 800<\/sup>C. In initial UV-visible spectrophotometric analysis, a single surface plasmon resonance peak between 420-450 nm with kmax at 440 nm was recorded in the observations which confirmed the formation of silver nanoparticles thereafter confirmed by TEM. The spherical shaped NFAgNPs were seen in TEM analysis. TEM images clearly demonstrated 20-50 nm size range of NFAgNPs. In addition, TEM micrograph revealed that most of the nanoparticles were spherical in shape. The biosynthesized NFAgNPs were evaluated for their free radical quenching ability by various in vitro<\/em> assays which exhibited higher DPPH radical scavenging activity as compared to ABTS in response to different concentrations of AgNPs. The free radical scavenging activity of NFAgNPs was found to increase significantly with increasing concentrations similar with observations noted in former some of the studies.13-14<\/sup> It is demonstrated earlier that the radical scavenging ability of biogenic silver nanoparticles is attributed due to integration of existed functional groups onto the surface of biogenic AgNPs originated from plant extract.15<\/sup> Earlier studies on in vitro<\/em> antioxidant potential of biosynthesized silver nanoparticles from Pongamia pinnata<\/em> showed considerable free radical scavenging potential.16<\/sup> Also, Kharat and Mendhulkar reported strong antioxidant activity in terms of DPPH radical scavenging by phytosynthesized silver nanoparticles using Elephantopus scaber.<\/em>17<\/sup> Similarly silver nanoparticles synthesized using plant extracts are significantly illustrated for their antioxidant potential.6-7<\/sup><\/p>\n

Recently several reports in the literature proved that many of the bacteria developed resistance to multiple antibiotics which ultimately leads necessity of alternate substitute for treatment of pathogenic microbes.18-19<\/sup> Several researchers developed alternate strategies to overcome the drug resistance. The biologically synthesized nanoparticles have emerged as a combatant to fight against microorganisms and overcome the dilemma of drug resistance. The enhanced antibacterial activity of biogenic nanoparticles is due to the synergistic effect between nanoparticles and natural compounds present in phytochemicals.20<\/sup> In our results we observed that the NFAgNPs exhibited promising antibacterial activity against booth Gram-positive and Gram negative bacteria. The extent of inhibitory effects on bacterial growth was observed on selected bacterial strains where E.coli<\/em> found to be more sensitive followed by P. aeruginosa<\/em> and K. pneumoniae<\/em> strains. Similar effects of biogenic silver nanoparticles synthesized using Eucalyptus globulus<\/em>21 <\/sup>and Murraya koeniggi <\/em>22<\/sup> are reported earlier. The biologically synthesized AgNPs using different plant extracts also showed a similar potent bactericidal activity.23-26<\/sup> Our results reported comparatively lower antibacterial activity of NFAgNPs against methicillin resistant Gram- positive S. aureus<\/em> bacteria in contrast to the results obtained by Ansari and M. A. Alzohairy27<\/sup>\u00a0with the biosynthesized silver nanoparticles using Phoenix dactylifera<\/em>\u00a0seed extracts. In conclusion, this is the first study investigate antioxidant and antibacterial effects of biogenic AgNPs synthesized by using N. foetida<\/em>. Our study also provide possible evidences supporting N. foetida<\/em> plant extract derived AgNPs exhibited strong antioxidant and antibacterial potential against selected bacterial strains.<\/p>\n

Acknowledgement<\/strong><\/p>\n

The authors gratefully acknowledge all the facilities and financial support provided by the Krishna Institute of Medical Sciences \u201cDeemed to be University\u201d Karad, India for experimental work. Authors are thankful to Mr. Santosh Jadhav & Mrs. Ashwini More for technical support.<\/p>\n

Conflict of Interest<\/strong><\/p>\n

There is no conflict of interest<\/p>\n

References<\/strong><\/p>\n

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