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Hydrothermal combustion based ZnO nanoparticles from Croton bonplandianum: Characterization and evaluation of antibacterial and antioxidant potential
Sustainable Chemistry and Pharmacy 14 (2019) 100186
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Sustainable Chemistry and Pharmacy journal homepage: http://www.elsevier.com/locate/scp
Hydrothermal combustion based ZnO nanoparticles from Croton bonplandianum: Characterization and evaluation of antibacterial and antioxidant potential N. Chandra Mohana a, C. Mahendra b, H.C. Yashavantha Rao c, M.R. Abhilash d, S. Satish a, * a
Bionanotechnological Laboratory, Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysore 570006, Karnataka, India Plant Tissue Culture and Biotechnology Laboratory, Department of Studies in Botany, University of Mysore, Manasagangotri, Mysuru 570 006, Karnataka, India Department of Biochemistry, Indian Institute of Science, Bengaluru 560012, Karnataka, India d Department of Studies in Environmental Science, University of Mysore, Manasagangotri, Mysuru 570 006, Karnataka, India b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Antibacterial Anti-oxidant Croton bonplandianum Bail ZnO NPs
The present study reports facile phytomediated synthesis of ZnO nanoparticles (ZnO NPs) using hydrothermal combustion from Croton bonplandianum Bail. The characterization studies revealed that an average 44 nm size spherical ZnO NPs with high zinc composition having good crystalline nature. FTIR analysis confirmed the presence of functional groups O–H, sp3 C–H bend, and alkoxy C–O functional groups which might be involved in capping and reduction for ZnO NPs synthesis. The biosynthesized ZnO NPs exhibited potent antibacterial activity against Gram-positive and Gram-negative bacteria. The MIC results revealed that ZnO NPs were most effective against Bacillus cereus whereas least sensitive towards Pseudomonas aeruginosa and Vibrio parahaemolyticus. This investigation suggests that biosynthesized ZnO NPs are promising agents for antibacterial and antioxidant ap plications in the biomedical field.
1. Introduction Nanotechnology has been under tremendous development over the past decades and applied in various fields of biomedicine. Among metal oxide nanoparticles, zinc oxide (ZnO) nanoparticles (NPs) have a broad range of applications like as sensors, semiconductors, resistors, optical, anti-corrosives, and piezoelectric properties along with biopotential ability (Jiang et al., 2018). Presently, ZnO has been employed in the biological fields for bio-imaging, biosensors, drug and gene delivery system (Das et al., 2013). It also has a wide range of applications as antimicrobials, antioxidants and anticancer agents in pharma field (Sruthi et al., 2018). The synthesis of nanoparticles using plant extracts has more advantages than the synthesis using any physical or chemical methods because of its economic and eco-friendly nature. They have also been opted for their high stability which increases their ability to syn thesize nanoparticles (Rajiv et al., 2013; Salam et al., 2014). There are recent reports of ZnO NPs synthesized from various plants such as Oci mum americanum, Malus pumila, Juglen regia, Ceropegia candelabrum and Cochlospermum religiosum and evaluated for various biological potential
(Stan et al., 2016; Murali et al., 2017; Mahendra et al., 2017; Kalpana and Devi Rajeswari, 2018). Croton bonplandianum Bail is an attribute of family Euphorbiaceae. In India, it is distributed in the tropical and subtropical regions of West Bengal. The plant is identified by different vernacular names: threeleaved caper (English), kalabhangre (Hindi), alpabedhi soppu (Kannada), eliamanakkau (Tamil), kukka mirapa (Telugu) (Chakrabarty and Balakrishnan, 1997; Rahman and Akter, 2013). Phytochemically C. bonplandianum is rich in secondary metabolites which comprises mainly alkaloids and terpenoids (Chandel et al., 1996). The different parts of this plant are used for treating various ailments and has bioactive potential like antioxidants, anti-cancer, antihypertensives, antimicrobials, antiseptics and nematicides (Singh et al., 2015). It is also reported to possess repellent activity against Aedes aegypti, antidote, analgesic, anthelminthic and larvicidal activities (Jeeshna et al., 2011). The present investigation reports on the biosynthesis of ZnO NPs synthesized from Croton bonplandianum Bail. owing to its ethno medicinal properties and its evaluation for antibacterial and antioxidant activities.
* Corresponding author. Bionanotechnological Laboratory, Department of Studies in Microbiology, Manasagangotri, University of Mysore, Mysore 570006, Kar nataka, India. E-mail address: [email protected] (S. Satish). https://doi.org/10.1016/j.scp.2019.100186 Received 28 July 2019; Received in revised form 10 October 2019; Accepted 12 October 2019 Available online 22 October 2019 2352-5541/© 2019 Elsevier B.V. All rights reserved.
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2. Materials and methods
2.4.2. Minimum inhibitory concentration (MIC) The MIC value for biosynthesized ZnO NPs was evaluated by micro broth dilution assay in 96 well plates (CLSI, 2012). The assay was carried out in two-fold dilution with a stock solution of ZnO-NPs (100 mg/mL 1), ZnO (5–0.002 mg mL 1) and standard gentamicin (5–0.002 mg mL 1). A predetermined inoculum suspension of 10 μL was added to each well in 100 μL of Muller Hinton Broth after treatment microtiter plates were incubated at 37 � 2 � C for 24 h, absorbance was measured at 620 nm using ELISA plate reader (LabTech 4000, Japan). Microbial growth indicator 2,3,5-triphenyl tetrazolium chloride (10 μL) was dispensed in each well followed by incubation for 30 min. The lowest concentrations with no visible growth were observed was taken as the MIC.
Chemicals: All the required chemicals were procured from HiMedia (Mumbai, India), Zinc nitrate hexahydrate (99%, SDFCL, Mumbai), Nanopure water, Sterile double distilled water, BHT (Butylated hydroxytoluene), Muller Hinton medium, Gentamicin. 2.1. Plant Collection and preparation of aqueous leaf extract Croton bonplandianum Bail Healthy asymptomatic leaves of Croton bonplandianum Bail. were collected from different locations around the University of Mysore, Mysuru, Karnataka, India and authenticated by Plant taxonomist. The collected plant leaf material was washed under tap water initially, fol lowed by double sterile distilled water twice for removing debris from the leaf surface. The blot dried leaf material (100 g) was ground using pestle and mortar, later mixed with 100 ml of nanopure water and allowed to boil for 1 h. The aqueous extract obtained was subjected to centrifuged at 10,000 rpm for 20 min, filtered thrice through Whatman filter paper No.1 and stored in 200 mL media flask.
2.4.3. Antioxidant assay 2.4.3.1. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. The DPPH radical scavenging activity of synthesized ZnO-NPs was determined in different concentrations range of 25, 50 and 100 μg mL 1. By using methanol, 0.1 mM of freshly prepared DPPH solution was mixed with each concentration of the tested sample and vortexed thoroughly. The reaction mixture was incubated at room temperature in dark for 20 min and absorbance at 517 nm was determined using UV-VIS spectroscopy (DU739, Beckman Coulter, Germany). BHT (Butylated hydroxytoluene) served as a positive control. The DPPH radical scav enging activity was expressed in terms of percentage inhibition using the following formula:
2.2. Synthesis of ZnO NPs The aqueous extract was subjected to phytogenic fabrication of ZnO NPs as described by Mahendra et al. (2017). The aqueous extract was dissolved in 2 g of Zinc nitrate hexahydrate, initially mixed using mag netic stirrer, upon solubilization the solution mixture was boiled at 60 � C until the solution becomes a paste. The paste obtained was subjected to a temperature of 300 � C for 2 h using ceramic crucible, the final white fine powdered product was used for characterization and biological assays.
% Scavenging effect¼((Ac–As))/Ac � 100 where Ac is the absorbance of the control, and As is the absorbance of the sample or standard.
2.3. Characterization of ZnO NPs
2.6. Statistical analysis
The optical characterization was carried out for preliminary confir mation of bioreduction in synthesized ZnO NPs using UV-VIS spectros copy (DU739, Beckman Coulter, Germany) with wavelength range 200–800 nm in 1 nm resolution (Gupta et al., 2014). The particle size and zeta potential along with their size distribution were determined by Dynamic Light Scattering (DLS) analysis (Microtrac, USA) (Kumar et al., 2019). The binding properties and functional groups involved in the formation of ZnO NPs was carried out by FTIR analysis in the mid-IR region of 400–4000 cm 1 using ATR technique (PerkinElmer Spectrum 1000) (Vanathi et al., 2014). The phase identity and crystallinity of ZnO NPs were characterized by X-ray diffraction analysis (Rigaku Desktop Miniflex II X-ray powder diffractometer). The Scanning electron microscopic imaging (SEM) (HITACHI S–3400 N, Japan) coupled with EDS (Hitachi Noran System 7, USA) operated at 10 kV acceleration voltages was employed for morphological and elemental composition of biosynthesized ZnO NPs followed by the method of Mahendra et al. (2017).
The data obtained from three independent replicate trials were analyzed for statistical significance using SPSS Inc. 25.0. The signifi cance of results is reported as mean � SD and significant differences between mean values were determined with for analysis of variance at Pvalue 0.001 followed by Tukey’s Post Hoc test with P-value 0.05. 3. Results and discussion 3.1. Characterization of biofabricated ZnO nanoparticles The bioreduced and stabilized ZnO-NPs formation was determined by optical characterization using UV–Vis spectroscopy (Fig. 1). The optical absorption spectra were measured by dispersing ZnO-NPs in water for the range of 200–800 nm. It is known that the optical ab sorption measurement for metal bounded nanoparticles is a well-known technique to characterize nanoparticles (Gupta et al., 2014). The ob tained nanoparticles showed excitation peak absorption at 290 nm indicated the presence of ZnO-NPs measured at room temperature. Ac cording to Mahendra et al. (2017) and Kumar et al. (2019), our results were correlated with their reports and it is also confirmed by many re searchers the excitation range between 280 and 330 nm indicate the characteristic feature of phyto-mediated ZnO NPs. The results obtained from dynamic light scattering (DLS) analysis is represented as marginal particle size distribution histograms which revealed an average size of 44 nm (Fig. 2). Our results are in concurrence with earlier reports of Vishnukumar et al. (2018) where the uniform dispersal nature indicates the purity and homogeneity of NPs. To determine the active functional groups responsible for the potential nature of the plant extracts for the synthesis of ZnO-NPs was performed. The FT-IR spectra for both plant extract and ZnO-NPs were determined with a resolution of 4 cm 1 with a wavelength range of 4000 to 600 cm 1 by ATR method. The FT-IR
2.4. Antibacterial activity 2.4.1. Antibacterial activity by disc diffusion assay The antibacterial susceptibility of ZnO NPs was evaluated against MTCC cultures Bacillus cereus (MTCC 430), Klebsiella pneumonia (MTCC 7407), Staphylococcus aureus (MTCC 7443), Vibrio parahaemolyticus (MTCC 451), Escherichia coli (MTCC 7410), Pseudomonas aeruginosa (MTCC 7903), Salmonella typhi (MTCC 733), Xanthomonas citri (MTCC 7908) by disc diffusion method (CLSI, 2009). Muller Hinton agar me dium pre-seeded with 106 test bacteria cultures using sterile cotton swabs were placed with sterile discs (6 mm) impregnated with bio synthesized ZnO NPs (50 μg/disc) along with Zinc nitrate (50 μg/disc) as negative control and positive control Gentamicin (10 μg/disc) followed by incubation at 37 � 2 � C and zone of inhibition was measured. 2
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spectrum comparison of both plant extract and biosynthesized ZnO NPs revealed possible reduction and capping involved in synthesis (Fig. 3). Broad peaks corresponding to 3495 cm 1, 3234 cm 1 and 3368 cm 1 showed the presence of O–H stretch. A strong absorption peak at – O. Peaks at 1683 cm 1 indicates that the stretching vibration due to C– 1433 cm 1 and 1401 cm 1 were observed corresponding to sp3 C–H bending. The peak 1054 cm 1 and 1030 cm 1 indicates the alkoxy C–O group. The FT-IR revealed synthesis and stabilization which might be due to O–H, sp3 C–H bend and alkoxy C–O functional groups involved in capping and reduction. These results matched with the existing report of Gnanasangeetha and Thambwani (2013) where they have reported the biosynthesis of ZnO nanoparticles using Acalypha indica leaf extract are clearly revealed the presence of active functional groups. The X-ray diffractometer spectrum, diffraction angles are 33.06� , 34.08� ,36.24� , 47.38� ,56.64� ,63.04� ,65.4� ,66.8� ,68.14� and 76.68� (Fig. 4) were corresponded to (100), (002), (101), (102), (110), (103), (200) and (112) planes matching JCPDS card 36–1451 for zinc oxide structure (Xia et al., 2008). The peaks observed in the XRD spectrum confirmed the high crystalline nature of biosynthesized ZnO NPs. The SEM-EDS analysis of ZnO NPs from Croton bonplandianum Bail. has been represented in Figs. 5 and 6. The biosynthesized ZnO NPs appeared to be spherical in shape with a pure phase of ZnO confirmed by EDS. The characterization studies confirmed the high purity ZnO NPs.
Fig. 1. UV–Vis spectra of synthesized ZnO NPs synthesized from Croton bon plandianum Bail.
3.1.1. Antibacterial activity The antibacterial activity of ZnO-NPs was performed by disk diffu sion assay to assess the efficacy of biosynthesized metal nanoparticle (Table 1; Fig. 7). The antibacterial test exhibited significant (p > 0.05) zone of inhibition against Staphylococcus aureus (26.67 mm); Pseudo monas aeruginosa (26.33 mm); Salmonella typhi (25.00 mm); Vibrio par ahaemolyticus (24.67 mm); Escherichia coli (22.00 mm); Xanthomonas citri (21.00 mm); Bacillus subtilis (19.67 mm) and Klebsiella pneumonia (15.67 mm). The results obtained in this study were validated with standard antibiotic Gentamicin (p > 0.05) as a positive control. Similar results were also reported by Lingaraju et al. (2016) and Bajpai et al. (2010), wherein the plant extracts mediated biosynthesized ZnO-NPs effectively inhibit both Gram-positive and Gram-negative bacterial strains. Likewise, there was no inhibition exerted by raw ZnO material which was also reported by Mahendra et al. (2017); Kumar et al. (2019) but potential antibacterial efficacy was observed in biofabricated ZnO NPs. The result clearly indicates the raw zinc material did not cause
Fig. 2. Marginal particle size of hydrothermal synthesized ZnO NPs from Croton bonplandianum Bail. by DLS analysis method.
Fig. 3. FTIR spectrum of synthesized ZnO NPs from Croton bonplandianum Bail. 3
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Fig. 4. X-ray diffraction spectrum of synthesized ZnO NPs from Croton bonplandianum Bail.
Fig. 5. Scanning electron microscopic analysis of synthesized ZnO NPs from Croton bonplandianum Bail.
Fig. 6. EDS analysis of synthesized ZnO NPs from Croton bonplandianum Bail.
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Table 1 Determination of antibacterial activity and minimum inhibitory concentrations (MIC) synthesized ZnO NPs from Croton bonplandianum Bail. by disc diffusion assay. Zone of inhibition measured in mm
MIC mg mL
1
Organism
ZnO NPs
Gentamicin
Control
ZnO NPs
Gentamicin
Control
Escherichia coli Salmonella typhi Pseudomonas aeruginosa Xanthomonas citri Bacillus cereus Klebsiella pneumonia Staphylococcus aureus Vibrio parahaemolyticus
22.00 � 1.00b 25.00 � 1.00a 26.33 � 0.58a 21.00 � 1.00b 19.67 � 0.58b 15.67 � 0.58c 26.67 � 0.58a 24.67 � 1.15a
21.33 � 0.58cd 28.33 � 0.58a 21.67 � 0.58cd 20.67 � 0.58d 25.33 � 0.58b 22.33 � 0.58c 25.33 � 0.58b 20.67 � 0.58d
NA NA NA NA NA NA NA NA
0.1562 0.3125 0.625 0.1562 0.0781 0.3125 0.1562 0.625
0.0195 0.0195 0.0195 0.0390 0.0097 0.0390 0.0195 0.0781
NA NA NA NA NA NA NA NA
Note: NA- No activity. Values followed by same superscript letter(s) are significantly different at (p<0.05) by Tukey’sPost Hoc Test.
Fig. 7. Evaluation of antibacterial activity of synthesized ZnO NPs from Croton bonplandianum Bail. by disc diffusion assay (ZnO NP- hydrothermal synthesized ZnO NPs, C- Zinc nitrate, G- Gentamicin).
toxicity against tested bacterium but ZnO-NPs allied with phytochemi cals can effectively damage the cell morphology which attributed to causing the membrane permeability which leads to cytoplasmic contents extrusions (Divyapriya et al., 2014).
Gram-negative bacterium. The mechanism of ZnO-NPs inhibition against bacteria is still not elucidated completely. However, some of the hypothesis proposed include disruption of cell permeability by direct interaction with cell wall/membrane by the release of metal ions (Xie et al., 2011). The other mechanisms include protein damage and DNA damage causing a disturbance in the metabolic process leading to cell death (Sirelkhatim et al., 2015).
3.1.2. Minimum inhibitory concentration (MIC) The drug which possesses antimicrobial property has minimum inhibitory dose concentrations against each specific bacterium. Like wise, the MIC value was evaluated for biosynthesized ZnO-NPs, ZnO and standard Gentamicin for MTCC test cultures and the corresponding MIC values are represented in Table 1. The biosynthesized ZnO-NPs was most potent against Bacillus cereus with MIC value of 0.0781 mg mL 1 whereas Escherichia coli, Xanthomonas citri and Staphylococcus aureus had a MIC of 0.1562 mg mL 1. The MIC value for Salmonella typhi and Klebsiella pneumonia was found to be 0.3125 mg mL 1 for ZnO-NPs treatments. For Pseudomonas aeruginosa and Vibrio parahaemolyticus MIC was observed at 0.625 mg mL 1and Gentamicin MIC value was found to be least against all the tested stains. The MIC results were in agreement with previous reports of Gunalan et al. (2012); Alekish et al., (2018); Siddiqi et al. (2018). The antibacterial susceptibility of ZnO-NPs has been found to be more against Gram-positive bacteria than
3.1.3. Antioxidant assay The comparative antioxidant activity of biofabricated ZnO NPs along with standard drug BHT was evaluated by DPPH method. The free radical scavenging antioxidant activity exerted by ZnO-NPs was found to be potent and the result was presented in Fig. 8. The significant increase in a free radical scavenging activity was observed with an increase in the concentration of ZnO-NPs. Our results are also in line with the earlier reports of Umar et al. (2019) wherein, the NPs synthesized from Albizia lebbeck shown the antioxidant property in a concentration-dependent manner. The percentage inhibition shown by synthesized nano particles is close to that of standard BHT, which could be due to zinc ions ligated with plant secondary metabolites. The biofabricated zinc oxide nano particles revealed the dose-dependent inhibition against free 5
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Fig. 8. Determination of anti-oxidant activity of synthesized ZnO NPs from Croton bonplandianum Bail.
radicals. It has been reported that phytochemical contents in green plants like phenols, alkaloids, steroids resins, etc. can utilize metal ions (Zn) as a cofactor resulting in the formation of inhibition of free radicals. Likewise, many researchers were also reported phenolic and other phytochemical present in the plant extracts influence high antioxidant capacity and they can play an important role in protecting cellular damages and other oxidative stress induced by reactive oxygen species (Singh et al., 2014; Parashant et al., 2015; Kokila et al., 2016).
Chandel, K.P.S., Shukla, G., Sharma, N., 1996. Biodiversity in Medicinal and Aromatic Plants in India. CLSI, C., 2009. Performance of Standards for Antimicrobial Disk Susceptibility Tests. Approved Standards. M2-A10. CLSI, 2012. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, approved standard. In: CLSI Document M07-A9, ninth ed. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania, USA. Das, S., Mitra, S., Khurana, S.M.P., Debnath, N., 2013. Nanomaterials for biomedical applications. Front. Life Sci. 7 (3–4), 90–98. Divyapriya, S., Sowmia, C., Sasikala, S., 2014. Synthesis of zinc oxide nanoparticles and antimicrobial activity of Murraya Koenigii. World J. Pharm. Pharm. Sci. 3 (12), 1635–1645. Gnanasangeetha, D., Thambwani, D.S., 2013. Biogenic production of zinc oxide nanoparticles using Acalypha indica. J. Chem. Biol. Phys. Sci. 1, 238–246. Gunalan, S., Sivaraj, R., Rajendran, V., 2012. Green synthesized ZnO nanoparticles against bacterial and fungal pathogens. Prog. Nat. Sci.: Materials International 22 (6), 693–700. Gupta, A., Srivastava, P., Bahadur, L., Amalnerkar, D.P., Chauhan, R., 2014. Comparison of physical and electrochemical properties of ZnO prepared via different surfactantassisted precipitation routes. Appl. Nanosci 5, 787–794. Jeeshna, M.V., Paulsamy, S., Mallikadevi, T., 2011. Phytochemical constituents and antimicrobial studies of the exotic plant species, Croton bonplandianum Baill. J. Life Sci. 3 (1), 23–27. Jiang, J., Pi, J., Cai, J., 2018. The advancing of zinc oxide nanoparticles for biomedical applications. Bioinorgan. Chem. Appl. 2018, 18. Article ID 1062562. Kalpana, V.N., Devi Rajeswari, V., 2018. A review on green synthesis, biomedical applications, and toxicity studies of ZnO NPs Bioinorgan. Chem. Appl. 2018, 3569758, 12 pages, 2018. https://doi.org/10.1155/2018/3569758. Kokila, T., Ramesh, P.S., Geetha, D., 2016. Biosynthesis of AgNPs using Carica papaya peel extract and evaluation of its antioxidant and antimicrobial activities. Ecotoxicol. Environ. Saf. 134 (Pt 2), 467–473. Kumar, H.N., Mohana, N.C., Nuthan, B.R., Ramesha, K.P., Rakshith, D., Geetha, N., Satish, S., 2019. Phyto-mediated synthesis of zinc oxide nanoparticles using aqueous plant extract of Ocimum americanum and evaluation of its bioactivity. SN Applied Sciences 1 (6), 651. Mahendra, C., Murali, M., Manasa, G., Pooja Ponnamma, Abhilash, M.R., Lakshmeesha, T.R., Satish, A., Amruthesh, K.N., Sudarshana, M.S., 2017. Antibacterial and antimitotic potential of bio-fabricated zinc oxide nanoparticles of Cochlospermum religiosum (L.). Microbial pathogenesis 110, 620–629. Murali, M., Mahendra, C., Rajashekar, N., Sudarshana, M.S., Raveesha, K.A., Amruthesh, K.N., 2017. Antibacterial and antioxidant properties of biosynthesized zinc oxide nanoparticles from Ceropegia candelabrum L.–an endemic species. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 179, 104–109. Lingaraju, K., Naika, H.R., Manjunath, K., Basavaraj, R.B., Nagabhushana, H., Nagaraju, G., Suresh, D., 2016. Biogenic synthesis of zinc oxide nanoparticles using Ruta graveolens (L.) and their antibacterial and antioxidant activities. Appl. Nanosci. 6 (5), 703–710. Parashant, G.K., Prashant, P.A., Utpal, B., et al., 2015. In vitro antibacterial and cytotoxicity studies of ZnO nanopowders prepared by combustion assisted facile green synthesis. Karbala Int J Mod Sci 1 (2), 67–77. Rahman, A.H.M.M., Akter, M., 2013. Taxonomy and medicinal uses of Euphorbiaceae (Spurge) family of Rajshahi, Bangladesh. Research in Plant Sciences 1 (3), 74–80. Rajiv, P., Rajeshwari, S., Venckatesh, R., 2013. Rambutan peels promoted biomimetic synthesis of bioinspired zinc oxide nanochains for biomedical applications. Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 112, 384–387.
4. Conclusion The present study reports phytogenic synthesis of ZnO NPs from Croton bonplandianum Bail. The biosynthesis of ZnO NPs as an easy, rapid, ecofriendly and economical method, when compared with phys ical and chemical methods, has been demonstrated. The phytofabricated ZnO NPs exhibited potent antimicrobial and antioxidant activity. Declaration of competing interest Authors declare no conflicts of interest. Acknowledgments The first author Chandra Mohana. N would like to thank ICMR, New Delhi for providing senior research fellowship (ICMR-SRF award No: 45/ 69/2018-PHA/BMS/OL). Mahendra C is thankful to the University of Mysore for Junior Research Fellowship (JRF) from SC/ST Cell (Uni. Order.No: ViGA: 04/11/2015-16, Dated: July 11, 2016). Dr. Yasha vantha Rao H.C. is thankful to University Grants Commission (UGC), New Delhi for the award of Dr. D.S. Kothari Post-Doctoral Fellowship award of Research Associate. The authors are grateful to University with Potential for Excellence (UPE) and Department of Studies in Microbi ology, University of Mysore, Mysuru for providing the necessary facilities. References Alekish, M., Ismail, Z.B., Albiss, B., Nawasrah, S., 2018. In vitro antibacterial effects of zinc oxide nanoparticles on multiple drug-resistant strains of Staphylococcus aureus and Escherichia coli: an alternative approach for antibacterial therapy of mastitis in sheep. Vet. World 11 (10), 1428. Bajpai, S.K., Chand, N., Chaurasia, V., 2010. Investigation of water vapor permeability and antimicrobial property of zinc oxide nanoparticles-loaded chitosan-based edible film. J. Appl. Polym. Sci. 115 (2), 674–683. Chakrabarty, T., Balakrishnan, N.P., 1997. A revision of Croton L.(Euphorbiaceae) for Indian subcontinent. Bull. Bot. Surv. India 34, 1–88.
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Sustainable Chemistry and Pharmacy journal homepage: http://www.elsevier.com/locate/scp
Hydrothermal combustion based ZnO nanoparticles from Croton bonplandianum: Characterization and evaluation of antibacterial and antioxidant potential N. Chandra Mohana a, C. Mahendra b, H.C. Yashavantha Rao c, M.R. Abhilash d, S. Satish a, * a
Bionanotechnological Laboratory, Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysore 570006, Karnataka, India Plant Tissue Culture and Biotechnology Laboratory, Department of Studies in Botany, University of Mysore, Manasagangotri, Mysuru 570 006, Karnataka, India Department of Biochemistry, Indian Institute of Science, Bengaluru 560012, Karnataka, India d Department of Studies in Environmental Science, University of Mysore, Manasagangotri, Mysuru 570 006, Karnataka, India b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Antibacterial Anti-oxidant Croton bonplandianum Bail ZnO NPs
The present study reports facile phytomediated synthesis of ZnO nanoparticles (ZnO NPs) using hydrothermal combustion from Croton bonplandianum Bail. The characterization studies revealed that an average 44 nm size spherical ZnO NPs with high zinc composition having good crystalline nature. FTIR analysis confirmed the presence of functional groups O–H, sp3 C–H bend, and alkoxy C–O functional groups which might be involved in capping and reduction for ZnO NPs synthesis. The biosynthesized ZnO NPs exhibited potent antibacterial activity against Gram-positive and Gram-negative bacteria. The MIC results revealed that ZnO NPs were most effective against Bacillus cereus whereas least sensitive towards Pseudomonas aeruginosa and Vibrio parahaemolyticus. This investigation suggests that biosynthesized ZnO NPs are promising agents for antibacterial and antioxidant ap plications in the biomedical field.
1. Introduction Nanotechnology has been under tremendous development over the past decades and applied in various fields of biomedicine. Among metal oxide nanoparticles, zinc oxide (ZnO) nanoparticles (NPs) have a broad range of applications like as sensors, semiconductors, resistors, optical, anti-corrosives, and piezoelectric properties along with biopotential ability (Jiang et al., 2018). Presently, ZnO has been employed in the biological fields for bio-imaging, biosensors, drug and gene delivery system (Das et al., 2013). It also has a wide range of applications as antimicrobials, antioxidants and anticancer agents in pharma field (Sruthi et al., 2018). The synthesis of nanoparticles using plant extracts has more advantages than the synthesis using any physical or chemical methods because of its economic and eco-friendly nature. They have also been opted for their high stability which increases their ability to syn thesize nanoparticles (Rajiv et al., 2013; Salam et al., 2014). There are recent reports of ZnO NPs synthesized from various plants such as Oci mum americanum, Malus pumila, Juglen regia, Ceropegia candelabrum and Cochlospermum religiosum and evaluated for various biological potential
(Stan et al., 2016; Murali et al., 2017; Mahendra et al., 2017; Kalpana and Devi Rajeswari, 2018). Croton bonplandianum Bail is an attribute of family Euphorbiaceae. In India, it is distributed in the tropical and subtropical regions of West Bengal. The plant is identified by different vernacular names: threeleaved caper (English), kalabhangre (Hindi), alpabedhi soppu (Kannada), eliamanakkau (Tamil), kukka mirapa (Telugu) (Chakrabarty and Balakrishnan, 1997; Rahman and Akter, 2013). Phytochemically C. bonplandianum is rich in secondary metabolites which comprises mainly alkaloids and terpenoids (Chandel et al., 1996). The different parts of this plant are used for treating various ailments and has bioactive potential like antioxidants, anti-cancer, antihypertensives, antimicrobials, antiseptics and nematicides (Singh et al., 2015). It is also reported to possess repellent activity against Aedes aegypti, antidote, analgesic, anthelminthic and larvicidal activities (Jeeshna et al., 2011). The present investigation reports on the biosynthesis of ZnO NPs synthesized from Croton bonplandianum Bail. owing to its ethno medicinal properties and its evaluation for antibacterial and antioxidant activities.
* Corresponding author. Bionanotechnological Laboratory, Department of Studies in Microbiology, Manasagangotri, University of Mysore, Mysore 570006, Kar nataka, India. E-mail address: [email protected] (S. Satish). https://doi.org/10.1016/j.scp.2019.100186 Received 28 July 2019; Received in revised form 10 October 2019; Accepted 12 October 2019 Available online 22 October 2019 2352-5541/© 2019 Elsevier B.V. All rights reserved.
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2. Materials and methods
2.4.2. Minimum inhibitory concentration (MIC) The MIC value for biosynthesized ZnO NPs was evaluated by micro broth dilution assay in 96 well plates (CLSI, 2012). The assay was carried out in two-fold dilution with a stock solution of ZnO-NPs (100 mg/mL 1), ZnO (5–0.002 mg mL 1) and standard gentamicin (5–0.002 mg mL 1). A predetermined inoculum suspension of 10 μL was added to each well in 100 μL of Muller Hinton Broth after treatment microtiter plates were incubated at 37 � 2 � C for 24 h, absorbance was measured at 620 nm using ELISA plate reader (LabTech 4000, Japan). Microbial growth indicator 2,3,5-triphenyl tetrazolium chloride (10 μL) was dispensed in each well followed by incubation for 30 min. The lowest concentrations with no visible growth were observed was taken as the MIC.
Chemicals: All the required chemicals were procured from HiMedia (Mumbai, India), Zinc nitrate hexahydrate (99%, SDFCL, Mumbai), Nanopure water, Sterile double distilled water, BHT (Butylated hydroxytoluene), Muller Hinton medium, Gentamicin. 2.1. Plant Collection and preparation of aqueous leaf extract Croton bonplandianum Bail Healthy asymptomatic leaves of Croton bonplandianum Bail. were collected from different locations around the University of Mysore, Mysuru, Karnataka, India and authenticated by Plant taxonomist. The collected plant leaf material was washed under tap water initially, fol lowed by double sterile distilled water twice for removing debris from the leaf surface. The blot dried leaf material (100 g) was ground using pestle and mortar, later mixed with 100 ml of nanopure water and allowed to boil for 1 h. The aqueous extract obtained was subjected to centrifuged at 10,000 rpm for 20 min, filtered thrice through Whatman filter paper No.1 and stored in 200 mL media flask.
2.4.3. Antioxidant assay 2.4.3.1. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. The DPPH radical scavenging activity of synthesized ZnO-NPs was determined in different concentrations range of 25, 50 and 100 μg mL 1. By using methanol, 0.1 mM of freshly prepared DPPH solution was mixed with each concentration of the tested sample and vortexed thoroughly. The reaction mixture was incubated at room temperature in dark for 20 min and absorbance at 517 nm was determined using UV-VIS spectroscopy (DU739, Beckman Coulter, Germany). BHT (Butylated hydroxytoluene) served as a positive control. The DPPH radical scav enging activity was expressed in terms of percentage inhibition using the following formula:
2.2. Synthesis of ZnO NPs The aqueous extract was subjected to phytogenic fabrication of ZnO NPs as described by Mahendra et al. (2017). The aqueous extract was dissolved in 2 g of Zinc nitrate hexahydrate, initially mixed using mag netic stirrer, upon solubilization the solution mixture was boiled at 60 � C until the solution becomes a paste. The paste obtained was subjected to a temperature of 300 � C for 2 h using ceramic crucible, the final white fine powdered product was used for characterization and biological assays.
% Scavenging effect¼((Ac–As))/Ac � 100 where Ac is the absorbance of the control, and As is the absorbance of the sample or standard.
2.3. Characterization of ZnO NPs
2.6. Statistical analysis
The optical characterization was carried out for preliminary confir mation of bioreduction in synthesized ZnO NPs using UV-VIS spectros copy (DU739, Beckman Coulter, Germany) with wavelength range 200–800 nm in 1 nm resolution (Gupta et al., 2014). The particle size and zeta potential along with their size distribution were determined by Dynamic Light Scattering (DLS) analysis (Microtrac, USA) (Kumar et al., 2019). The binding properties and functional groups involved in the formation of ZnO NPs was carried out by FTIR analysis in the mid-IR region of 400–4000 cm 1 using ATR technique (PerkinElmer Spectrum 1000) (Vanathi et al., 2014). The phase identity and crystallinity of ZnO NPs were characterized by X-ray diffraction analysis (Rigaku Desktop Miniflex II X-ray powder diffractometer). The Scanning electron microscopic imaging (SEM) (HITACHI S–3400 N, Japan) coupled with EDS (Hitachi Noran System 7, USA) operated at 10 kV acceleration voltages was employed for morphological and elemental composition of biosynthesized ZnO NPs followed by the method of Mahendra et al. (2017).
The data obtained from three independent replicate trials were analyzed for statistical significance using SPSS Inc. 25.0. The signifi cance of results is reported as mean � SD and significant differences between mean values were determined with for analysis of variance at Pvalue 0.001 followed by Tukey’s Post Hoc test with P-value 0.05. 3. Results and discussion 3.1. Characterization of biofabricated ZnO nanoparticles The bioreduced and stabilized ZnO-NPs formation was determined by optical characterization using UV–Vis spectroscopy (Fig. 1). The optical absorption spectra were measured by dispersing ZnO-NPs in water for the range of 200–800 nm. It is known that the optical ab sorption measurement for metal bounded nanoparticles is a well-known technique to characterize nanoparticles (Gupta et al., 2014). The ob tained nanoparticles showed excitation peak absorption at 290 nm indicated the presence of ZnO-NPs measured at room temperature. Ac cording to Mahendra et al. (2017) and Kumar et al. (2019), our results were correlated with their reports and it is also confirmed by many re searchers the excitation range between 280 and 330 nm indicate the characteristic feature of phyto-mediated ZnO NPs. The results obtained from dynamic light scattering (DLS) analysis is represented as marginal particle size distribution histograms which revealed an average size of 44 nm (Fig. 2). Our results are in concurrence with earlier reports of Vishnukumar et al. (2018) where the uniform dispersal nature indicates the purity and homogeneity of NPs. To determine the active functional groups responsible for the potential nature of the plant extracts for the synthesis of ZnO-NPs was performed. The FT-IR spectra for both plant extract and ZnO-NPs were determined with a resolution of 4 cm 1 with a wavelength range of 4000 to 600 cm 1 by ATR method. The FT-IR
2.4. Antibacterial activity 2.4.1. Antibacterial activity by disc diffusion assay The antibacterial susceptibility of ZnO NPs was evaluated against MTCC cultures Bacillus cereus (MTCC 430), Klebsiella pneumonia (MTCC 7407), Staphylococcus aureus (MTCC 7443), Vibrio parahaemolyticus (MTCC 451), Escherichia coli (MTCC 7410), Pseudomonas aeruginosa (MTCC 7903), Salmonella typhi (MTCC 733), Xanthomonas citri (MTCC 7908) by disc diffusion method (CLSI, 2009). Muller Hinton agar me dium pre-seeded with 106 test bacteria cultures using sterile cotton swabs were placed with sterile discs (6 mm) impregnated with bio synthesized ZnO NPs (50 μg/disc) along with Zinc nitrate (50 μg/disc) as negative control and positive control Gentamicin (10 μg/disc) followed by incubation at 37 � 2 � C and zone of inhibition was measured. 2
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spectrum comparison of both plant extract and biosynthesized ZnO NPs revealed possible reduction and capping involved in synthesis (Fig. 3). Broad peaks corresponding to 3495 cm 1, 3234 cm 1 and 3368 cm 1 showed the presence of O–H stretch. A strong absorption peak at – O. Peaks at 1683 cm 1 indicates that the stretching vibration due to C– 1433 cm 1 and 1401 cm 1 were observed corresponding to sp3 C–H bending. The peak 1054 cm 1 and 1030 cm 1 indicates the alkoxy C–O group. The FT-IR revealed synthesis and stabilization which might be due to O–H, sp3 C–H bend and alkoxy C–O functional groups involved in capping and reduction. These results matched with the existing report of Gnanasangeetha and Thambwani (2013) where they have reported the biosynthesis of ZnO nanoparticles using Acalypha indica leaf extract are clearly revealed the presence of active functional groups. The X-ray diffractometer spectrum, diffraction angles are 33.06� , 34.08� ,36.24� , 47.38� ,56.64� ,63.04� ,65.4� ,66.8� ,68.14� and 76.68� (Fig. 4) were corresponded to (100), (002), (101), (102), (110), (103), (200) and (112) planes matching JCPDS card 36–1451 for zinc oxide structure (Xia et al., 2008). The peaks observed in the XRD spectrum confirmed the high crystalline nature of biosynthesized ZnO NPs. The SEM-EDS analysis of ZnO NPs from Croton bonplandianum Bail. has been represented in Figs. 5 and 6. The biosynthesized ZnO NPs appeared to be spherical in shape with a pure phase of ZnO confirmed by EDS. The characterization studies confirmed the high purity ZnO NPs.
Fig. 1. UV–Vis spectra of synthesized ZnO NPs synthesized from Croton bon plandianum Bail.
3.1.1. Antibacterial activity The antibacterial activity of ZnO-NPs was performed by disk diffu sion assay to assess the efficacy of biosynthesized metal nanoparticle (Table 1; Fig. 7). The antibacterial test exhibited significant (p > 0.05) zone of inhibition against Staphylococcus aureus (26.67 mm); Pseudo monas aeruginosa (26.33 mm); Salmonella typhi (25.00 mm); Vibrio par ahaemolyticus (24.67 mm); Escherichia coli (22.00 mm); Xanthomonas citri (21.00 mm); Bacillus subtilis (19.67 mm) and Klebsiella pneumonia (15.67 mm). The results obtained in this study were validated with standard antibiotic Gentamicin (p > 0.05) as a positive control. Similar results were also reported by Lingaraju et al. (2016) and Bajpai et al. (2010), wherein the plant extracts mediated biosynthesized ZnO-NPs effectively inhibit both Gram-positive and Gram-negative bacterial strains. Likewise, there was no inhibition exerted by raw ZnO material which was also reported by Mahendra et al. (2017); Kumar et al. (2019) but potential antibacterial efficacy was observed in biofabricated ZnO NPs. The result clearly indicates the raw zinc material did not cause
Fig. 2. Marginal particle size of hydrothermal synthesized ZnO NPs from Croton bonplandianum Bail. by DLS analysis method.
Fig. 3. FTIR spectrum of synthesized ZnO NPs from Croton bonplandianum Bail. 3
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Fig. 4. X-ray diffraction spectrum of synthesized ZnO NPs from Croton bonplandianum Bail.
Fig. 5. Scanning electron microscopic analysis of synthesized ZnO NPs from Croton bonplandianum Bail.
Fig. 6. EDS analysis of synthesized ZnO NPs from Croton bonplandianum Bail.
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Table 1 Determination of antibacterial activity and minimum inhibitory concentrations (MIC) synthesized ZnO NPs from Croton bonplandianum Bail. by disc diffusion assay. Zone of inhibition measured in mm
MIC mg mL
1
Organism
ZnO NPs
Gentamicin
Control
ZnO NPs
Gentamicin
Control
Escherichia coli Salmonella typhi Pseudomonas aeruginosa Xanthomonas citri Bacillus cereus Klebsiella pneumonia Staphylococcus aureus Vibrio parahaemolyticus
22.00 � 1.00b 25.00 � 1.00a 26.33 � 0.58a 21.00 � 1.00b 19.67 � 0.58b 15.67 � 0.58c 26.67 � 0.58a 24.67 � 1.15a
21.33 � 0.58cd 28.33 � 0.58a 21.67 � 0.58cd 20.67 � 0.58d 25.33 � 0.58b 22.33 � 0.58c 25.33 � 0.58b 20.67 � 0.58d
NA NA NA NA NA NA NA NA
0.1562 0.3125 0.625 0.1562 0.0781 0.3125 0.1562 0.625
0.0195 0.0195 0.0195 0.0390 0.0097 0.0390 0.0195 0.0781
NA NA NA NA NA NA NA NA
Note: NA- No activity. Values followed by same superscript letter(s) are significantly different at (p<0.05) by Tukey’sPost Hoc Test.
Fig. 7. Evaluation of antibacterial activity of synthesized ZnO NPs from Croton bonplandianum Bail. by disc diffusion assay (ZnO NP- hydrothermal synthesized ZnO NPs, C- Zinc nitrate, G- Gentamicin).
toxicity against tested bacterium but ZnO-NPs allied with phytochemi cals can effectively damage the cell morphology which attributed to causing the membrane permeability which leads to cytoplasmic contents extrusions (Divyapriya et al., 2014).
Gram-negative bacterium. The mechanism of ZnO-NPs inhibition against bacteria is still not elucidated completely. However, some of the hypothesis proposed include disruption of cell permeability by direct interaction with cell wall/membrane by the release of metal ions (Xie et al., 2011). The other mechanisms include protein damage and DNA damage causing a disturbance in the metabolic process leading to cell death (Sirelkhatim et al., 2015).
3.1.2. Minimum inhibitory concentration (MIC) The drug which possesses antimicrobial property has minimum inhibitory dose concentrations against each specific bacterium. Like wise, the MIC value was evaluated for biosynthesized ZnO-NPs, ZnO and standard Gentamicin for MTCC test cultures and the corresponding MIC values are represented in Table 1. The biosynthesized ZnO-NPs was most potent against Bacillus cereus with MIC value of 0.0781 mg mL 1 whereas Escherichia coli, Xanthomonas citri and Staphylococcus aureus had a MIC of 0.1562 mg mL 1. The MIC value for Salmonella typhi and Klebsiella pneumonia was found to be 0.3125 mg mL 1 for ZnO-NPs treatments. For Pseudomonas aeruginosa and Vibrio parahaemolyticus MIC was observed at 0.625 mg mL 1and Gentamicin MIC value was found to be least against all the tested stains. The MIC results were in agreement with previous reports of Gunalan et al. (2012); Alekish et al., (2018); Siddiqi et al. (2018). The antibacterial susceptibility of ZnO-NPs has been found to be more against Gram-positive bacteria than
3.1.3. Antioxidant assay The comparative antioxidant activity of biofabricated ZnO NPs along with standard drug BHT was evaluated by DPPH method. The free radical scavenging antioxidant activity exerted by ZnO-NPs was found to be potent and the result was presented in Fig. 8. The significant increase in a free radical scavenging activity was observed with an increase in the concentration of ZnO-NPs. Our results are also in line with the earlier reports of Umar et al. (2019) wherein, the NPs synthesized from Albizia lebbeck shown the antioxidant property in a concentration-dependent manner. The percentage inhibition shown by synthesized nano particles is close to that of standard BHT, which could be due to zinc ions ligated with plant secondary metabolites. The biofabricated zinc oxide nano particles revealed the dose-dependent inhibition against free 5
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Fig. 8. Determination of anti-oxidant activity of synthesized ZnO NPs from Croton bonplandianum Bail.
radicals. It has been reported that phytochemical contents in green plants like phenols, alkaloids, steroids resins, etc. can utilize metal ions (Zn) as a cofactor resulting in the formation of inhibition of free radicals. Likewise, many researchers were also reported phenolic and other phytochemical present in the plant extracts influence high antioxidant capacity and they can play an important role in protecting cellular damages and other oxidative stress induced by reactive oxygen species (Singh et al., 2014; Parashant et al., 2015; Kokila et al., 2016).
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4. Conclusion The present study reports phytogenic synthesis of ZnO NPs from Croton bonplandianum Bail. The biosynthesis of ZnO NPs as an easy, rapid, ecofriendly and economical method, when compared with phys ical and chemical methods, has been demonstrated. The phytofabricated ZnO NPs exhibited potent antimicrobial and antioxidant activity. Declaration of competing interest Authors declare no conflicts of interest. Acknowledgments The first author Chandra Mohana. N would like to thank ICMR, New Delhi for providing senior research fellowship (ICMR-SRF award No: 45/ 69/2018-PHA/BMS/OL). Mahendra C is thankful to the University of Mysore for Junior Research Fellowship (JRF) from SC/ST Cell (Uni. Order.No: ViGA: 04/11/2015-16, Dated: July 11, 2016). Dr. Yasha vantha Rao H.C. is thankful to University Grants Commission (UGC), New Delhi for the award of Dr. D.S. Kothari Post-Doctoral Fellowship award of Research Associate. The authors are grateful to University with Potential for Excellence (UPE) and Department of Studies in Microbi ology, University of Mysore, Mysuru for providing the necessary facilities. References Alekish, M., Ismail, Z.B., Albiss, B., Nawasrah, S., 2018. In vitro antibacterial effects of zinc oxide nanoparticles on multiple drug-resistant strains of Staphylococcus aureus and Escherichia coli: an alternative approach for antibacterial therapy of mastitis in sheep. Vet. World 11 (10), 1428. Bajpai, S.K., Chand, N., Chaurasia, V., 2010. Investigation of water vapor permeability and antimicrobial property of zinc oxide nanoparticles-loaded chitosan-based edible film. J. Appl. Polym. Sci. 115 (2), 674–683. Chakrabarty, T., Balakrishnan, N.P., 1997. A revision of Croton L.(Euphorbiaceae) for Indian subcontinent. Bull. Bot. Surv. India 34, 1–88.
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