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Green fabrication, characterization of Pisonia alba leaf extract derived MgO nanoparticles and its biological applications
Nano-Structures & Nano-Objects 20 (2019) 100380
Contents lists available at ScienceDirect
Nano-Structures & Nano-Objects journal homepage: www.elsevier.com/locate/nanoso
Green fabrication, characterization of Pisonia alba leaf extract derived MgO nanoparticles and its biological applications ∗
Govindasamy Sharmila a , , Chandrasekaran Muthukumaran a , Elango Sangeetha a , Harikrishnan Saraswathi a , Selvaraj Soundarya a , Narasimhan Manoj Kumar b a b
Department of Industrial Biotechnology, Government College of Technology, Coimbatore 641013, Tamil Nadu, India Department of Genetic Engineering, SRM Institute of Science & Technology, Kattankulathur 603203, Tamil Nadu, India
highlights
graphical
abstract
• First report on green synthesis of MgO NPs using Pisonia alba leaf extract. • P. alba leaf extract derived MgO NPs showed good antioxidant activity. • MgO NPs exhibited strong fungicidal activity against A. flavus and F. solani.
article
info
Article history: Received 30 July 2019 Accepted 2 August 2019 Keywords: Magnesium oxide Nanoparticles Pisonia alba Antioxidant Antifungal
a b s t r a c t A facile, eco-friendly green synthesis of magnesium oxide nanoparticles (MONPs) using Pisonia alba leaf extract was reported. The MONPs were characterized by UV–Vis, TEM, EDX, XRD and FTIR. A good antioxidant activity was exhibited by P. alba leaf extract derived MONPs assessed by DPPH and FRAP assays. Antifungal activity assay results revealed that Aspergillus flavus and Fusarium solani were highly inhibited by green synthesized MONPs. The results of this study demonstrated that P. alba leaf extract derived MONPs showed good antioxidant, antifungal properties and it can be utilized for biomedical and food applications. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Nanotechnology is a science which utilizes various techniques to synthesize the nanoparticles with larger surface area possessing unique behaviour and special characteristics [1]. Nanoparticles with such special properties and distinctive behaviour can vastly influence the biological, physico-chemical, electrical and mechanical function of any system [2]. In recent years, nanotechnology provided its extensive contribution in the fields of medical, biomedical, agriculture, automobiles, electronics, drug delivery, packaging, cosmetics, green chemistry and various bioengineering [3–5]. Synthesis of metallic nanoparticles opens up new pathway in the field of nanotechnology as it possesses additional special features when compare with other nanoparticles. Among metallic nanoparticles, extensive research has been ∗ Corresponding author. E-mail address: [email protected] (G. Sharmila). https://doi.org/10.1016/j.nanoso.2019.100380 2352-507X/© 2019 Elsevier B.V. All rights reserved.
carried out using gold, silver nanoparticles and its applications in agriculture, electronics and biomedical field as drug delivery system, anti-microbial, and anti-cancer agents [6,7]. Even though many different metallic nanoparticles has been utilized in various fields of research, magnesium oxide nanoparticles (MONPs) attracted more researchers due to its non-toxic nature and costeffective. Food and Drug Administration (FDA) recommended MONPs as safe materials and hence substantial research attempts were made in recent years due to its extensive applications in medical fields. Earlier investigations on MONPs reported that it possesses excellent bactericidal activity against Staphylococcus aureus, E. coli [8] and against aggressive plant pathogen such as Ralstonia solanacearum [9]. There are different methods to synthesize MONPs which includes: sol–gel process [10], co-precipitation [11], microwave, hydrothermal [12], sonochemical [13] etc. Presently, green synthesis of metal nanoparticles using plant extract gains much attention since it is eco-friendly and cost-effective [14–16]. MONPs synthesis via green route is
2
G. Sharmila, C. Muthukumaran, E. Sangeetha et al. / Nano-Structures & Nano-Objects 20 (2019) 100380
limited in literature and few studies on MONPs green synthesis using Limonia acidissima [17], Andrographis paniculata [18], Costus pictus [19], Artemisia abrotanum [20], Nephelium lappaceum peels [21], gum acacia [22] and neem leaves [23] were reported previously. In this study, Pisonia alba leaf extract mediated green synthesis of MONPs was investigated for first time. P. alba is a well-known elaborate plant belongs to Nyctaginaceae family and grown well in India, Sri Lanka and Philippines. The oblongovate shaped leaves are oppositely arranged in 20 cm length. This plant contains various bioactive compounds such as allantoin, sitosterol, quercetin, dulcitol and pinnatol. It is used for several medical applications since it possess anticancer, larvicidal and ovicidal properties [24–26]. The present study focused on the biosynthesis of MONPs using P. alba leaf extract. The synthesized MONPs were characterized by UV–Vis spectroscopy, TEM, EDX, XRD and FTIR analysis. Further, antioxidant and antifungal applications of MONPs was also reported in this study.
and 50% methanol was used as blank [26]. The scavenging activity of MONPs was calculated using Eq. (1) Scavenging activity (%)
=
(absorbance of the control − absorbance of sample) absorbance of control
× 100 (1)
In FRAP assay, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine), 300 mM acetate buffer (3.1 g sodium acetate trihydrate and 16 mL acetic acid, pH 3.6) and 20 mM ferricchloride hexahydrate in 40 mM HCl were mixed to prepare FRAP mixture. 50 mL of acetate buffer and 5 ml of TPTZ solution in 5 mL ferric chloride hexahydrate warmed at 37 ◦ C was used as working standard. MONPs (2– 10 mg) was added to 4 ml of FRAP reagent in different test tubes and mixed well. The reaction mixture was incubated in dark place for 30 min and the absorbance were measured for the coloured product (ferrous tripridyltriazine complex) at 593 nm in the UV–Vis spectrophotometer.
2. Materials and methods 2.5. Antifungal activity of MONPs 2.1. Chemicals Analytical grade chemicals, Magnesium nitrate, DPPH (2,2diphenyl-1-picrylhydrazyl), Potato dextrose agar, Fluconazole, Ferric chloride and TPTZ (2,4,6-tripyridyl-s-triazine) were purchased from Hi-Media. 2.2. Green synthesis of MONPs using P. alba leaf extract Fresh Pisonia alba leaves were collected from Arni, Thiruvannamalai district, Tamil Nadu and the leaves were cleaned with tap water, shade dried and powdered. 5 g of the leaf powder was added to 100 mL of distilled water and kept in water bath for 15 min at 70 ◦ C [26]. The mixture was stirred using a magnetic stirrer for about 20 min and extract was collected by filtering the solution using the Whatman paper. 1 mM Mg(NO3 )2 precursor solution and P. alba leaf extract were mixed in 4:1 ratio and the mixture was incubated at room temperature for complete synthesis of MONPs. The formation of MONPs was observed by measuring the absorbance in the wavelength range of 200–700 nm using UV–Vis spectrophotometer (Perkin Elmer) at regular intervals, After completion of MONPs synthesis, the solution was centrifuged at 5000 rpm for 10 min, dried at 80 ◦ C and stored for further studies. 2.3. Characterization of MONPs The morphological characterization of green synthesized MONPs was done with Transmission electron microscopy (FEI, Tecnai G2 model). The elemental profile of the MONPs was identified by EDX detector attached with the TEM instrument. The X’Pert pro X-ray Diffractometer was used to study the crystallinity of MONPs. FT-IR (Perkin Elmer) analysis was performed to determine the chemical bonds present in MONPs surface using KBr pellet method in the scan range of 4000–450 cm−1 .
The two fungal strains, Aspergillus flavus and Fusarium solani were chosen as test organisms for the fungicidal activity of MONPs. Agar well-diffusion method was adopted and potato dextrose agar medium (3.9 g in 100 mL) was used. The sterile PDA medium was prepared and added to sterilized petriplate for solidification. 150 µL of the corresponding fungal spores were spread with sterile cotton swab and six wells (5 mm) were made with cork borer. MONPs (25–100 µg/mL) concentrations were added to the wells and incubated at 29 ◦ C for 5 days. Fluconazole and distilled water were used as positive and negative control respectively. After incubation, the zone of inhibition (ZOI) was calculated for each MONPs concentration. 3. Results and discussion 3.1. UV–Vis spectroscopy UV–Visible absorption spectroscopy is most extensively used to characterize the optical properties of as-synthesized nanoparticles [20]. In this study, the visual transition of colour change from pale yellow to dark brown indicates the formation of phytosynthesized MONPS mediated by P.alba which was further confirmed by UV–Vis absorption spectroscopy. The appearance of surface plasmon resonance (SPR) band at shorter wavelengths below 300 nm specifies the existence of small sized particles [27]. The SPR peak obtained at 272 nm which falls in the range of 260 and 280 nm confirmed the formation of phytosynthesized nanoparticles as MONPs (Fig. 1). The obtained SPR peak confirmed the phytoreduction of Mg (NO3)2 to MONPS and it was clear that the phytochemicals present in the P. alba leaf may function as a capping and stabilizing agent towards the phytosynthesis of MONPS. The obtained results were well concordant with the previous literature reported the absorption peak at 273 nm for MONPs synthesized by neem leaves via green route [23].
2.4. Antioxidant activity 3.2. TEM and EDX analysis The DPPH (2,2-diphenyl-1-picrylhydrazyl) and Ferric reducing antioxidant power (FRAP) assays were performed to estimate the antioxidant capacity of MONPs synthesized using P. alba leaf extract. In DPPH assay, MONPs (2–10 mg) was added to 2.5 mL of 50% methanol and well mixed. 140 µL of DPPH reagent (0.14 mM) was added to each test tube and kept in dark for 30 min. After incubation, the absorbance was measured at 517 nm
TEM analysis of MONPs showed roughly spherical shape and the average particle size was found to be less than 100 nm (Fig. 2a) and in accordance with the results reported by Raliya et al. [28]. EDX spectrum represented in Fig. 2b confirmed the characteristic peak signals due to the presence of elements such as oxygen (O), magnesium (Mg) for the as-synthesized MONPs
G. Sharmila, C. Muthukumaran, E. Sangeetha et al. / Nano-Structures & Nano-Objects 20 (2019) 100380
Fig. 1. UV–vis spectrum of green synthesized MONPs using P. alba leaf extract.
which was in agreement with the previous report [29]. Dobruka et al. reported that the morphology of the phytosynthesized MONPs using the aqueous extract of Artemisia abrotanum herb was spherical shape through TEM analysis [20]. EDS profile showed the evidence for the formation of MgO nanoparticles. Cai et al. also reported the size of the roughly spherical MONPs was in the range of 50–100 nm [9]. 3.3. XRD analysis X-ray diffraction pattern of the synthesized MONPs using P. alba leaf extract was shown in Fig. 3 and it displayed a face centred cubic crystalline phase corresponds to the crystal planes at 111, 220, 400 matches with JCPDS No: 002-1207. The diffraction pattern contains the diffraction peaks for cubic MgO correspond to crystal planes of (111), (200), (220), (311) and (222) was previously reported by Sharma et al. for MONPs synthesized using aqueous plant extract of Swertia chirayaita [30]. 3.4. FTIR analysis FTIR spectroscopy was used to identify the respective chemical groups present in the synthesized MONPs of P. alba leaf extract (Fig. 4). The band at 3290 cm−1 was assigned to O–H group
3
Fig. 3. XRD pattern of P. alba leaf extract derived MONPs.
which may belong to water or plant phenolic compounds. The occurrence of peak at 2928 cm−1 confirmed C–H group in MONPs. The bands at 1654 cm−1 and 1328 cm−1 attributed to (C==O) and (C–N) of amide and amine group respectively which confirm the presence of protein. The bending vibration at 959 cm−1 belongs to ==C–H bond of alkene group. The band observed in the range between 536–772 cm−1 attributed to Mg–O vibrations which confirmed Mg–O bond in the synthesized nanoparticles [31]. The chemical bonds (O–H), (C==O) and (C–N) identified in the FTIR analysis demonstrated that the phenolic compounds or proteins present in P. alba leaf extract may act as bioreducing agent for MONPs synthesis. 3.5. Antioxidant activity The antioxidant activity of the P.alba leaf extract derived MONPs assessed by DPPH radical scavenging and FRAP assay. For both DPPH and FRAP assay, the MONPs (2–10 mg) was taken and assessed for its free radical scavenging activity and reducing power activity. Maximum scavenging activity of 65% was observed at 4 mg and beyond increasing the concentration does not shows the increasing effect (Fig. 5a). The scavenging activity
Fig. 2. (a) TEM image and (b) EDX profile of green synthesized MONPs using P. alba leaf extract.
4
G. Sharmila, C. Muthukumaran, E. Sangeetha et al. / Nano-Structures & Nano-Objects 20 (2019) 100380
Fig. 4. FTIR spectrum of P. alba leaf extract derived MONPs.
observed may be due to the presence of biologically active components present in the P. alba leaf extract. The maximum reducing power activity of 69.3% observed at 10 mg of the MONPs and it is evident that increasing the concentration of MONPs increases the reducing power activity (Fig. 5b). Similar results on antioxidant studies of MONPs by DPPH radical scavenging and FRAP assay were reported by Sushma et al. [32]. 3.6. Antifungal activity The fungicidal activity of green synthesized MONPs was tested against two fungal strains Aspergillus flavus and Fusarium solani. The zone of inhibition (ZOI) at the concentrations of 75 mg/ml and 100 mg/ml were 2 mm and 4 mm respectively for A. flavus (Fig. 6a). Similarly, the ZOI of 2 mm and 3 mm was observed for F. solani at the concentrations of 75 mg/ml and 100 mg/ml respectively (Fig. 6b). On increasing concentration of MONPs, ZOI also increases resulting in higher antifungal activity. Generally, the antimicrobial activity by the synthesized MONPs may be due to the generation of reactive oxygen species (ROS) [33]. Pugazhendhi et al. [27] examined antifungal activity of the prepared MONPs
Fig. 5. (a) DPPH and (b) FRAP antioxidant activity of green synthesized MONPs using P. alba leaf extract.
against Aspergillus fumigates, Fusarium solani and Aspergillus niger with positive control (Fluconozole). It was reported that among all the three tested strains, MONPs was effective against Fusarium solani and Aspergillus niger than Aspergillus fumigates. Previous literature studies revealed that the shape of the nanoparticles also can influence the antimicrobial activity [34]. However, the
Fig. 6. Antifungal activity of green synthesized MONPs using P. alba leaf extract. (a) A. flavus and (b) F. solani.
G. Sharmila, C. Muthukumaran, E. Sangeetha et al. / Nano-Structures & Nano-Objects 20 (2019) 100380
information about the antifungal activity of MONPs was still limited. The possible mechanism of antifungal behaviour of MONPs was due to the electrostatic interaction between the MONPs and cell membrane proteins. The captured MONPs seems to be tightly adsorbed on to the microorganisms which generate ROS inside the fungal cell causes oxidative stress and leads to cell death [19]. 4. Conclusion A greener eco-friendly synthesis of MONPs using Pisonia alba leaf extract was reported. The synthesized MONPs were characterized by UV–Vis spectroscopy, TEM, EDX, XRD and FTIR analysis. In UV–Vis spectroscopy, SPR peak observed at 272 nm confirms the formation of MONPs. TEM analysis revealed that the size of synthesized MONPs was found to be less than 100 nm and EDX spectrum confirmed the presence of magnesium (Mg). Face centred cubic structure of MONPs was elucidated by XRD analysis. Functional groups responsible for the bioreduction and stabilization of the synthesized MONPs using the extract of P. alba were identified in FTIR analysis. The results of DPPH and FRAP assays revealed that the P. alba leaf extract derived MONPs showed good antioxidant activity. Antifungal activity results showed that MONPs possess strong fungicidal action against Aspergillus flavus and Fusarium solani. In conclusion, P.alba leaf extract derived MONPs can be used as an effective antioxidant and fungicidal agent in food packing, medical and agricultural applications. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] M.A. Usmani, I. Khan, A.H. Bhat, R.S. Pillai, N. Ahmad, M.K.M. Haafiz, M. Oves, Current trend in the application of nanoparticles for waste water treatment and purification: a review, Curr. Org. Synth. 14 (2017) 206–226. [2] S.S. Mukhopadhyay, Nanotechnology in agriculture: prospects and constraints, Nanotechnol. Sci. Appl. 7 (2014) 63. [3] R. Prasad, A. Bhattacharyya, Q.D. Nguyen, Nanotechnology in sustainable agriculture: recent developments, challenges, and perspectives, Front. Microbiol. 8 (2017) 1014. [4] M. Ghaani, N. Nasirizadeh, S.A.Y. Ardakani, F.Z. Mehrjardi, M. Scampicchio, S. Farris, Development of an electrochemical nanosensor for the determination of gallic acid in food, Anal. Method 8 (2016) 1103–1110. [5] R.G. Saratale, I. Karuppusamy, G.D. Saratale, A. Pugazhendhi, G. Kumar, Y. Park, G.S. Ghodake, R.N. Bhargava, J.R. Banu, H.S. Shin, A comprehensive review on green nanomaterials using biological systems: Recent perception and their future applications, Colloids Surf. B 170 (2018) 20–35. [6] W. Chen, Q. Zhang, B.L. Kaplan, G.L. Baker, N.E. Kaminski, Induced T cell cytokine production is enhanced by engineered nanoparticles, Nanotoxicol. 8 (2014) 11–23. [7] M. Oves, M. Aslam, M.A. Rauf, S. Qayyum, H.A. Qari, M.S. Khan, M.Z. Alam, S. Tabrez, A. Pugazhendhi, I.M. Ismail, Antimicrobial and anticancer activities of silver nanoparticles synthesized from the root hair extract of Phoenix dactylifera, Mater. Sci. Eng. C 89 (2018) 429–443. [8] L. Umaralikhan, M.J.M. Jaffar, Green synthesis of MgO nanoparticles and it antibacterial activity, Iran. J. Sci. Technol. Trans. A 42 (2018) 477–485. [9] L. Cai, J. Chen, Z. Liu, H. Wang, H. Yang, W. Ding, Magnesium oxide nanoparticles: effective agricultural antibacterial agent against Ralstonia solanacearum, Front. Microbiol. 9 (2018) 790. [10] O. Darčanova, M. Tamute, A. Beganskiene, A. Kareiva, Synthesis of magnesium oxide nanoparticles via sol–gel method and hydrolysis and application for paper deacidification treatment, Chemija 27 (2016) 170–178.
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Contents lists available at ScienceDirect
Nano-Structures & Nano-Objects journal homepage: www.elsevier.com/locate/nanoso
Green fabrication, characterization of Pisonia alba leaf extract derived MgO nanoparticles and its biological applications ∗
Govindasamy Sharmila a , , Chandrasekaran Muthukumaran a , Elango Sangeetha a , Harikrishnan Saraswathi a , Selvaraj Soundarya a , Narasimhan Manoj Kumar b a b
Department of Industrial Biotechnology, Government College of Technology, Coimbatore 641013, Tamil Nadu, India Department of Genetic Engineering, SRM Institute of Science & Technology, Kattankulathur 603203, Tamil Nadu, India
highlights
graphical
abstract
• First report on green synthesis of MgO NPs using Pisonia alba leaf extract. • P. alba leaf extract derived MgO NPs showed good antioxidant activity. • MgO NPs exhibited strong fungicidal activity against A. flavus and F. solani.
article
info
Article history: Received 30 July 2019 Accepted 2 August 2019 Keywords: Magnesium oxide Nanoparticles Pisonia alba Antioxidant Antifungal
a b s t r a c t A facile, eco-friendly green synthesis of magnesium oxide nanoparticles (MONPs) using Pisonia alba leaf extract was reported. The MONPs were characterized by UV–Vis, TEM, EDX, XRD and FTIR. A good antioxidant activity was exhibited by P. alba leaf extract derived MONPs assessed by DPPH and FRAP assays. Antifungal activity assay results revealed that Aspergillus flavus and Fusarium solani were highly inhibited by green synthesized MONPs. The results of this study demonstrated that P. alba leaf extract derived MONPs showed good antioxidant, antifungal properties and it can be utilized for biomedical and food applications. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Nanotechnology is a science which utilizes various techniques to synthesize the nanoparticles with larger surface area possessing unique behaviour and special characteristics [1]. Nanoparticles with such special properties and distinctive behaviour can vastly influence the biological, physico-chemical, electrical and mechanical function of any system [2]. In recent years, nanotechnology provided its extensive contribution in the fields of medical, biomedical, agriculture, automobiles, electronics, drug delivery, packaging, cosmetics, green chemistry and various bioengineering [3–5]. Synthesis of metallic nanoparticles opens up new pathway in the field of nanotechnology as it possesses additional special features when compare with other nanoparticles. Among metallic nanoparticles, extensive research has been ∗ Corresponding author. E-mail address: [email protected] (G. Sharmila). https://doi.org/10.1016/j.nanoso.2019.100380 2352-507X/© 2019 Elsevier B.V. All rights reserved.
carried out using gold, silver nanoparticles and its applications in agriculture, electronics and biomedical field as drug delivery system, anti-microbial, and anti-cancer agents [6,7]. Even though many different metallic nanoparticles has been utilized in various fields of research, magnesium oxide nanoparticles (MONPs) attracted more researchers due to its non-toxic nature and costeffective. Food and Drug Administration (FDA) recommended MONPs as safe materials and hence substantial research attempts were made in recent years due to its extensive applications in medical fields. Earlier investigations on MONPs reported that it possesses excellent bactericidal activity against Staphylococcus aureus, E. coli [8] and against aggressive plant pathogen such as Ralstonia solanacearum [9]. There are different methods to synthesize MONPs which includes: sol–gel process [10], co-precipitation [11], microwave, hydrothermal [12], sonochemical [13] etc. Presently, green synthesis of metal nanoparticles using plant extract gains much attention since it is eco-friendly and cost-effective [14–16]. MONPs synthesis via green route is
2
G. Sharmila, C. Muthukumaran, E. Sangeetha et al. / Nano-Structures & Nano-Objects 20 (2019) 100380
limited in literature and few studies on MONPs green synthesis using Limonia acidissima [17], Andrographis paniculata [18], Costus pictus [19], Artemisia abrotanum [20], Nephelium lappaceum peels [21], gum acacia [22] and neem leaves [23] were reported previously. In this study, Pisonia alba leaf extract mediated green synthesis of MONPs was investigated for first time. P. alba is a well-known elaborate plant belongs to Nyctaginaceae family and grown well in India, Sri Lanka and Philippines. The oblongovate shaped leaves are oppositely arranged in 20 cm length. This plant contains various bioactive compounds such as allantoin, sitosterol, quercetin, dulcitol and pinnatol. It is used for several medical applications since it possess anticancer, larvicidal and ovicidal properties [24–26]. The present study focused on the biosynthesis of MONPs using P. alba leaf extract. The synthesized MONPs were characterized by UV–Vis spectroscopy, TEM, EDX, XRD and FTIR analysis. Further, antioxidant and antifungal applications of MONPs was also reported in this study.
and 50% methanol was used as blank [26]. The scavenging activity of MONPs was calculated using Eq. (1) Scavenging activity (%)
=
(absorbance of the control − absorbance of sample) absorbance of control
× 100 (1)
In FRAP assay, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine), 300 mM acetate buffer (3.1 g sodium acetate trihydrate and 16 mL acetic acid, pH 3.6) and 20 mM ferricchloride hexahydrate in 40 mM HCl were mixed to prepare FRAP mixture. 50 mL of acetate buffer and 5 ml of TPTZ solution in 5 mL ferric chloride hexahydrate warmed at 37 ◦ C was used as working standard. MONPs (2– 10 mg) was added to 4 ml of FRAP reagent in different test tubes and mixed well. The reaction mixture was incubated in dark place for 30 min and the absorbance were measured for the coloured product (ferrous tripridyltriazine complex) at 593 nm in the UV–Vis spectrophotometer.
2. Materials and methods 2.5. Antifungal activity of MONPs 2.1. Chemicals Analytical grade chemicals, Magnesium nitrate, DPPH (2,2diphenyl-1-picrylhydrazyl), Potato dextrose agar, Fluconazole, Ferric chloride and TPTZ (2,4,6-tripyridyl-s-triazine) were purchased from Hi-Media. 2.2. Green synthesis of MONPs using P. alba leaf extract Fresh Pisonia alba leaves were collected from Arni, Thiruvannamalai district, Tamil Nadu and the leaves were cleaned with tap water, shade dried and powdered. 5 g of the leaf powder was added to 100 mL of distilled water and kept in water bath for 15 min at 70 ◦ C [26]. The mixture was stirred using a magnetic stirrer for about 20 min and extract was collected by filtering the solution using the Whatman paper. 1 mM Mg(NO3 )2 precursor solution and P. alba leaf extract were mixed in 4:1 ratio and the mixture was incubated at room temperature for complete synthesis of MONPs. The formation of MONPs was observed by measuring the absorbance in the wavelength range of 200–700 nm using UV–Vis spectrophotometer (Perkin Elmer) at regular intervals, After completion of MONPs synthesis, the solution was centrifuged at 5000 rpm for 10 min, dried at 80 ◦ C and stored for further studies. 2.3. Characterization of MONPs The morphological characterization of green synthesized MONPs was done with Transmission electron microscopy (FEI, Tecnai G2 model). The elemental profile of the MONPs was identified by EDX detector attached with the TEM instrument. The X’Pert pro X-ray Diffractometer was used to study the crystallinity of MONPs. FT-IR (Perkin Elmer) analysis was performed to determine the chemical bonds present in MONPs surface using KBr pellet method in the scan range of 4000–450 cm−1 .
The two fungal strains, Aspergillus flavus and Fusarium solani were chosen as test organisms for the fungicidal activity of MONPs. Agar well-diffusion method was adopted and potato dextrose agar medium (3.9 g in 100 mL) was used. The sterile PDA medium was prepared and added to sterilized petriplate for solidification. 150 µL of the corresponding fungal spores were spread with sterile cotton swab and six wells (5 mm) were made with cork borer. MONPs (25–100 µg/mL) concentrations were added to the wells and incubated at 29 ◦ C for 5 days. Fluconazole and distilled water were used as positive and negative control respectively. After incubation, the zone of inhibition (ZOI) was calculated for each MONPs concentration. 3. Results and discussion 3.1. UV–Vis spectroscopy UV–Visible absorption spectroscopy is most extensively used to characterize the optical properties of as-synthesized nanoparticles [20]. In this study, the visual transition of colour change from pale yellow to dark brown indicates the formation of phytosynthesized MONPS mediated by P.alba which was further confirmed by UV–Vis absorption spectroscopy. The appearance of surface plasmon resonance (SPR) band at shorter wavelengths below 300 nm specifies the existence of small sized particles [27]. The SPR peak obtained at 272 nm which falls in the range of 260 and 280 nm confirmed the formation of phytosynthesized nanoparticles as MONPs (Fig. 1). The obtained SPR peak confirmed the phytoreduction of Mg (NO3)2 to MONPS and it was clear that the phytochemicals present in the P. alba leaf may function as a capping and stabilizing agent towards the phytosynthesis of MONPS. The obtained results were well concordant with the previous literature reported the absorption peak at 273 nm for MONPs synthesized by neem leaves via green route [23].
2.4. Antioxidant activity 3.2. TEM and EDX analysis The DPPH (2,2-diphenyl-1-picrylhydrazyl) and Ferric reducing antioxidant power (FRAP) assays were performed to estimate the antioxidant capacity of MONPs synthesized using P. alba leaf extract. In DPPH assay, MONPs (2–10 mg) was added to 2.5 mL of 50% methanol and well mixed. 140 µL of DPPH reagent (0.14 mM) was added to each test tube and kept in dark for 30 min. After incubation, the absorbance was measured at 517 nm
TEM analysis of MONPs showed roughly spherical shape and the average particle size was found to be less than 100 nm (Fig. 2a) and in accordance with the results reported by Raliya et al. [28]. EDX spectrum represented in Fig. 2b confirmed the characteristic peak signals due to the presence of elements such as oxygen (O), magnesium (Mg) for the as-synthesized MONPs
G. Sharmila, C. Muthukumaran, E. Sangeetha et al. / Nano-Structures & Nano-Objects 20 (2019) 100380
Fig. 1. UV–vis spectrum of green synthesized MONPs using P. alba leaf extract.
which was in agreement with the previous report [29]. Dobruka et al. reported that the morphology of the phytosynthesized MONPs using the aqueous extract of Artemisia abrotanum herb was spherical shape through TEM analysis [20]. EDS profile showed the evidence for the formation of MgO nanoparticles. Cai et al. also reported the size of the roughly spherical MONPs was in the range of 50–100 nm [9]. 3.3. XRD analysis X-ray diffraction pattern of the synthesized MONPs using P. alba leaf extract was shown in Fig. 3 and it displayed a face centred cubic crystalline phase corresponds to the crystal planes at 111, 220, 400 matches with JCPDS No: 002-1207. The diffraction pattern contains the diffraction peaks for cubic MgO correspond to crystal planes of (111), (200), (220), (311) and (222) was previously reported by Sharma et al. for MONPs synthesized using aqueous plant extract of Swertia chirayaita [30]. 3.4. FTIR analysis FTIR spectroscopy was used to identify the respective chemical groups present in the synthesized MONPs of P. alba leaf extract (Fig. 4). The band at 3290 cm−1 was assigned to O–H group
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Fig. 3. XRD pattern of P. alba leaf extract derived MONPs.
which may belong to water or plant phenolic compounds. The occurrence of peak at 2928 cm−1 confirmed C–H group in MONPs. The bands at 1654 cm−1 and 1328 cm−1 attributed to (C==O) and (C–N) of amide and amine group respectively which confirm the presence of protein. The bending vibration at 959 cm−1 belongs to ==C–H bond of alkene group. The band observed in the range between 536–772 cm−1 attributed to Mg–O vibrations which confirmed Mg–O bond in the synthesized nanoparticles [31]. The chemical bonds (O–H), (C==O) and (C–N) identified in the FTIR analysis demonstrated that the phenolic compounds or proteins present in P. alba leaf extract may act as bioreducing agent for MONPs synthesis. 3.5. Antioxidant activity The antioxidant activity of the P.alba leaf extract derived MONPs assessed by DPPH radical scavenging and FRAP assay. For both DPPH and FRAP assay, the MONPs (2–10 mg) was taken and assessed for its free radical scavenging activity and reducing power activity. Maximum scavenging activity of 65% was observed at 4 mg and beyond increasing the concentration does not shows the increasing effect (Fig. 5a). The scavenging activity
Fig. 2. (a) TEM image and (b) EDX profile of green synthesized MONPs using P. alba leaf extract.
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G. Sharmila, C. Muthukumaran, E. Sangeetha et al. / Nano-Structures & Nano-Objects 20 (2019) 100380
Fig. 4. FTIR spectrum of P. alba leaf extract derived MONPs.
observed may be due to the presence of biologically active components present in the P. alba leaf extract. The maximum reducing power activity of 69.3% observed at 10 mg of the MONPs and it is evident that increasing the concentration of MONPs increases the reducing power activity (Fig. 5b). Similar results on antioxidant studies of MONPs by DPPH radical scavenging and FRAP assay were reported by Sushma et al. [32]. 3.6. Antifungal activity The fungicidal activity of green synthesized MONPs was tested against two fungal strains Aspergillus flavus and Fusarium solani. The zone of inhibition (ZOI) at the concentrations of 75 mg/ml and 100 mg/ml were 2 mm and 4 mm respectively for A. flavus (Fig. 6a). Similarly, the ZOI of 2 mm and 3 mm was observed for F. solani at the concentrations of 75 mg/ml and 100 mg/ml respectively (Fig. 6b). On increasing concentration of MONPs, ZOI also increases resulting in higher antifungal activity. Generally, the antimicrobial activity by the synthesized MONPs may be due to the generation of reactive oxygen species (ROS) [33]. Pugazhendhi et al. [27] examined antifungal activity of the prepared MONPs
Fig. 5. (a) DPPH and (b) FRAP antioxidant activity of green synthesized MONPs using P. alba leaf extract.
against Aspergillus fumigates, Fusarium solani and Aspergillus niger with positive control (Fluconozole). It was reported that among all the three tested strains, MONPs was effective against Fusarium solani and Aspergillus niger than Aspergillus fumigates. Previous literature studies revealed that the shape of the nanoparticles also can influence the antimicrobial activity [34]. However, the
Fig. 6. Antifungal activity of green synthesized MONPs using P. alba leaf extract. (a) A. flavus and (b) F. solani.
G. Sharmila, C. Muthukumaran, E. Sangeetha et al. / Nano-Structures & Nano-Objects 20 (2019) 100380
information about the antifungal activity of MONPs was still limited. The possible mechanism of antifungal behaviour of MONPs was due to the electrostatic interaction between the MONPs and cell membrane proteins. The captured MONPs seems to be tightly adsorbed on to the microorganisms which generate ROS inside the fungal cell causes oxidative stress and leads to cell death [19]. 4. Conclusion A greener eco-friendly synthesis of MONPs using Pisonia alba leaf extract was reported. The synthesized MONPs were characterized by UV–Vis spectroscopy, TEM, EDX, XRD and FTIR analysis. In UV–Vis spectroscopy, SPR peak observed at 272 nm confirms the formation of MONPs. TEM analysis revealed that the size of synthesized MONPs was found to be less than 100 nm and EDX spectrum confirmed the presence of magnesium (Mg). Face centred cubic structure of MONPs was elucidated by XRD analysis. Functional groups responsible for the bioreduction and stabilization of the synthesized MONPs using the extract of P. alba were identified in FTIR analysis. The results of DPPH and FRAP assays revealed that the P. alba leaf extract derived MONPs showed good antioxidant activity. Antifungal activity results showed that MONPs possess strong fungicidal action against Aspergillus flavus and Fusarium solani. In conclusion, P.alba leaf extract derived MONPs can be used as an effective antioxidant and fungicidal agent in food packing, medical and agricultural applications. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] M.A. Usmani, I. Khan, A.H. Bhat, R.S. Pillai, N. Ahmad, M.K.M. Haafiz, M. Oves, Current trend in the application of nanoparticles for waste water treatment and purification: a review, Curr. Org. Synth. 14 (2017) 206–226. [2] S.S. Mukhopadhyay, Nanotechnology in agriculture: prospects and constraints, Nanotechnol. Sci. Appl. 7 (2014) 63. [3] R. Prasad, A. Bhattacharyya, Q.D. Nguyen, Nanotechnology in sustainable agriculture: recent developments, challenges, and perspectives, Front. Microbiol. 8 (2017) 1014. [4] M. Ghaani, N. Nasirizadeh, S.A.Y. Ardakani, F.Z. Mehrjardi, M. Scampicchio, S. Farris, Development of an electrochemical nanosensor for the determination of gallic acid in food, Anal. Method 8 (2016) 1103–1110. [5] R.G. Saratale, I. Karuppusamy, G.D. Saratale, A. Pugazhendhi, G. Kumar, Y. Park, G.S. Ghodake, R.N. Bhargava, J.R. Banu, H.S. Shin, A comprehensive review on green nanomaterials using biological systems: Recent perception and their future applications, Colloids Surf. B 170 (2018) 20–35. [6] W. Chen, Q. Zhang, B.L. Kaplan, G.L. Baker, N.E. Kaminski, Induced T cell cytokine production is enhanced by engineered nanoparticles, Nanotoxicol. 8 (2014) 11–23. [7] M. Oves, M. Aslam, M.A. Rauf, S. Qayyum, H.A. Qari, M.S. Khan, M.Z. Alam, S. Tabrez, A. Pugazhendhi, I.M. Ismail, Antimicrobial and anticancer activities of silver nanoparticles synthesized from the root hair extract of Phoenix dactylifera, Mater. Sci. Eng. C 89 (2018) 429–443. [8] L. Umaralikhan, M.J.M. Jaffar, Green synthesis of MgO nanoparticles and it antibacterial activity, Iran. J. Sci. Technol. Trans. A 42 (2018) 477–485. [9] L. Cai, J. Chen, Z. Liu, H. Wang, H. Yang, W. Ding, Magnesium oxide nanoparticles: effective agricultural antibacterial agent against Ralstonia solanacearum, Front. Microbiol. 9 (2018) 790. [10] O. Darčanova, M. Tamute, A. Beganskiene, A. Kareiva, Synthesis of magnesium oxide nanoparticles via sol–gel method and hydrolysis and application for paper deacidification treatment, Chemija 27 (2016) 170–178.
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