Bioinspired green synthesis of copper oxide nanoparticles from Syzygium alternifolium (Wt.) Walp: characterization and evaluation of its synergistic antimicrobial and anticancer activity
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In recent times, nanoparticles are attributed to green nanotechnology methods to know the synergistic biological activities. To accomplish this phenomenon, present study was aimed to synthesize copper oxide nanoparticles (CuO NPs) by using Syzygium alternifolium stem bark, characterized those NPs using expository tools and to elucidate high prioritized antimicrobial and anticancer activities. Synthesized particles exhibited a color change pattern upon synthesis and affirmed its respective broad peak at 285 nm which was analyzed through UV–vis spectroscopy. FT-IR study confirmed that phenols and primary amines were mainly involved in capping and stabilization of nanoparticles. DLS and Zeta potential studies revealed narrow size of particles with greater stability. XRD studies revealed the crystallographic nature of particles with 17.2 nm average size. Microscopic analysis by using TEM revealed that particle size range from 5–13 nm and most of them were spherical in shape, non-agglomerated and poly-dispersed in condition. Antimicrobial studies of particles showed highest inhibitory activity against E. coli and T. harzianum among bacterial and fungal strains, respectively. The scope of this study is extended by examining anticancer activity of CuO NPs. This study exhibited potential anticancer activity towards MDA-MB-231 human breast cancer lines. Overall, these examinations relate that the S. alternifolium is described as efficient well-being plant and probabilistically for the design and synthesis of nanoparticles for human health. This study paves a way to better understand antimicrobial and anticancer therapeutic drug potentials of nanoparticles to design and analysis of pharmaceuticals by in vivo and in vitro approaches.
KeywordsSyzygium alternifolium Stem bark CuO NPs Characterization Antimicrobial activity Anticancer activity
In the present century research on nanotechnology gains much importance in the fields of biology, medicine, physics, chemistry and electronics. When coming to the biology nanoparticles are synthesized with the help of medicinal plants as reducing agents and is a fascinating research area by synthesizing different types of nanoparticles like, calcium, copper, gold, iron, silver and zinc (Kumar et al. 2016). Among these, copper nanoparticles have enormous considerations in the preparation of algaecides, fungicides, antifouling and antimicrobial agents. Thereafter which attract attention in various fields due to its catalytic, electric, optical, photonic and superconducting properties (Padil and Cernik 2013). The green synthesis method of CuO NPs with medicinal plants is eco-friendly, cost effective and stable. However, the research going on with this method is very seldom. Whereas the chemical- and physical-mediated methods were very familiar, but they arise some difficulties to humans as well as to the environment. In chemical-mediated synthesis, the copper salts were reduced with the help of reducing agents like, sodium borohydride, hydrazine, microemulsions and with evaporation/condensation methods. The use of sodium borohydride can irritate skin, eyes, nose and throat while exposure. Higher exposures may cause pulmonary edema and affect the nervous system (Yugandhar et al. 2015). Hydrazine is a colorless, flammable inorganic compound, exposure to this chemical cause irritation of eyes, nose and throat. Higher exposures may lead to seizure, coma, kidney and liver damages. The disadvantage of microemulsion was its high expensive cost, which may adsorb on the surface of particles, separation and removal of them from final products was difficult, which ultimately reduces the usability of nanoparticles (Capek 2004). The evaporation/condensation method occupies large space, consumes more power and the obtained powders were in highly agglomerated state. These nano-powders were treated with high purity gas streams to cause compositional and structural modifications of nanoparticles, which arises hazardous waste materials to the environment (Tavakoli et al. 2007).
Coming to the physical-mediated familiar methods, the pulse laser ablation, microwave-assisted and pulsed/explosion wire discharge methods are in trend. The disadvantage of the laser ablation method was its high cost, high energy consumption and low product efficiency (Kalyanaraman et al. 1998). The microwave-assisted synthesis has radiation health hazards and the cost of microwave systems. The pulsed/explosion wire discharge method was not cost effective, limited production efficiency and contaminations of the final product were considered as limitations to this method (Umer et al. 2012). With these constraints, medicinal plants are employed for the alternative and future strategies to synthesize nanoparticles. These medicinal plants are acting as eco-friendly, safety, cost effective and green reducing sources for synthesis of copper nanoparticles. In recent past, the CuO NPs were synthesized from Acalypha indica (Sivaraj et al. 2014), Aloe vera (Vijay Kumar et al. 2015) and Punica granatum (Ghidana et al. 2016). These nanoparticles exhibit different biological activities like, anticancer (Sankar et al. 2014), anti-inflammatory, mosquito larvicidal (Angajala et al. 2014), wound healing (Tiwari et al. 2014), antimicrobial (Shende et al. 2015), anti diabetic and antioxidant activity (Ghosh et al. 2015). However, there is no report on green synthesis of CuO NPs from S. alternifolium and there is no validation of antimicrobial activity against microorganisms and anticancer activity against MDA-MB-231 breast cancer cell lines. Hence, the present work has been undertaken to synthesize CuO NPs and to study their synergistic antimicrobial and anticancer activities.
The plant S. alternifolium was grown on top hills of Tirumala forest, Chittoor District, Andhra Pradesh, India. It belongs to the family Myrtaceae and is locally known as mogi or adavinerudu. This is a medium sized perennial tree and has importance in both commercial and medicinal practices. The plant was exploited in a commercial way for constructing buildings in the local premises. Fruit part of the plant was eaten as an edible fruit, cooked to make into squashes, jellies and local vinegar by the tribal people. Besides to these commercial purposes, the local people of Tirumala hills practiced to cure stomach ulcers by oral administration of fruit juice, external application of fruit pulp for rheumatic pains. Chenchu and Nakkala tribes of Tirumala hills were traditionally practiced to cure diarrhea by using fruit powder (Savithramma et al. 2014a). Yanadi tribe and local villagers of Veyilingalakona Sacred Grove utilized fruit powder to control diabetes (Savithramma et al. 2014b). The oral administration of stem bark decoction regulates the blood sugar levels and reduces intestinal ulcers (Sudhakar et al. 2012). Due to the exploited collection of plant parts for commercial as well as medicinal purposes and other biotic/abiotic baroreceptor factors, this plant was categorized under endemic and endangered species to these hills by “The IUCN Red List of Threatened Species” (Saha et al. 2015).
Materials and methods
Collection of plant material
Fresh part of S. alternifolium stem bark was collected from Nagatheertham area of Tirumala hills, Chittoor District, Andhra Pradesh, India. Stem bark was washed thrice with running tap water followed by Milli Pak pure water. They may dry up to 15–20 days under shady conditions to evaporate residual moisture and finally ground with blender for further use.
Deionized Milli Q water (Merck Water Solutions, France), CuSO4·5H2O (Sisco Research Laboratories Pvt. Ltd., India), NaOH, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT), Nutrient broth agar and Potato dextrose agar media (Sigma Aldrich, Bangalore, India), Fluconazole, Streptomycin and Doxorubicin (Thermo Fisher Scientifics, India) were mainly used in this work.
Synthesis of nanoparticles
5 g of finely grounded plant powder was extracted with 100 ml of Milli Q water on boiling water bath for 30 min and were filtered with Whatmann no. 1 filter paper. An aliquot of 10 ml of aqueous plant extract was titrated with 100 ml of 5 mM CuSO4·5H2O for reduction of CuO NPs at 50 °C for 2 h. Obtained mixture was centrifuged at 10,000 RPM for 15 min to separate agglomerated, broad sized particles as well as plant admixtures.
The green synthesized nanoparticles were characterized by using different spectroscopic and microscopic tools. Initial confirmation of nanoparticles was done by UV–vis spectroscopy (Spectro UV 2080, Analytical Technologies, India) to know which of the metal nanoparticles were actually reduced and which of the phytochemicals were actually involved in reduction of nanoparticles by surface plasmon resonance method. To comprehend which of the phytochemicals were actually involved in capping and stabilization of nanoparticles, the FTIR spectroscopy (ALPHA interferometer, ECO-ATR, Bruker, Ettlingen, Karlsruhe, Germany) was utilized. Dynamic light scattering (DLS) and Zeta potential of synthesized nanoparticles were analyzed to know the average size and stability of particles (Nanopartica analyzer, Horiba SZ 100, Japan). XRD (Shimadzu, XRD-6000) was used to analyze crystalline nature and calculate the average size of particles. Microscopic analysis with TEM (HF-3300, 300 kV TEM/STEM, Hitachi) instrument reveals the size, shape, dispersed nature and agglomerated pattern of nanoparticles. Along with these, the publicly accessed software tools like ImageJ and CrysTBox were used to know the average size and diffraction pattern of nanoparticles.
The antimicrobial activity of green synthesized nanoparticles was carried out against seven selected bacterial strains include Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922, Klebsiella pneumonia ATCC 43816, Proteus vulgaris ATCC 13315, Pseudomonas aeruginosa ATCC 15442, Salmonella typhimurium ATCC 14028 and five fungal strains include Alternaria solani ATCC 32904, Aspergillus flavus ATCC 9643, Aspergillus niger ATCC 16404, Penicillium chrysogenum ATCC 11709 and Trichoderma harzianum ATCC 20476 by using disc diffusion assay method (Cruickshank 1986). To know the minimum inhibitory concentration of prepared nanoparticles, serial concentrations of 5, 10, 20, 40 and 80 µg/ml were tested. Finally, found that 20 µg/ml concentrations showed minimum growth inhibition and 80 µg/ml concentrations as almost lethal toxic to all the microorganisms. For this, 40 µg/ml concentrations were used as LD50 value for antimicrobial activity of prepared nanoaprticles. The plant extract (1 mg/ml) and CuSO4·5H2O (5 mM) was used as positive and negative controls, respectively. Streptomycin and Fluconazole (10 µg/ml/disc) were used as standard controls for bacterial and fungal strains, respectively. An aliquot of 20 µl per disc was prepared for above-mentioned aliquots and an amount of 50 µl per petri plate microbial broth cultures were used for swabbing on culture medium. The triplicates of all these experiments were carried out under sterile conditions and were incubated at 37 °C for 24–48 h.
Results and discussion
After the reduction of CuO material, they formed as spherical shaped CuO nanoparticles by the action of plant phytochemicals. Plant phytochemicals act as capping and stabilizing agents to give particular shape and to avoid agglomeration between the particles (Baoshun et al. 2016). When analyzed these samples with UV–vis spectrophotometer between the scan range of 190–750 nm. Nanoparticles in the reaction mixture were excited by absorbing light at different wavelengths due to surface plasmon resonance (SPR) nature to give respective broad peak, represents which metal nanoparticles was actually reduced. Synthesized reaction mixture showed that a broad peak at 285 nm corresponds to copper oxide, which confirms the synthesized nanoparticles were CuO NPs. Another small peak obtained at 541 nm confirmed as ascorbic acid, acts as reducing agent for CuO NPs (Fig. 1). Similar type of result was observed from copper nanoparticles synthesized with the help of Punica granatum peel extract (Ghidana et al. 2016).
Zeta potential and particle size determination
The present study, reported a cost effective, eco-friendly, green approach method for production of CuO NPs from stem bark extract of S. alternifolium. Due to constraints with chemical- and physical-mediated methods, the green approach method is best opted technique to synthesize CuO NPs. The analytical tools like UV–vis spectroscopy and FT-IR studies revealed that the reduction of nanoparticle by ascorbic acid and the presence of phytochemicals like phenols and primary amines of proteins become helpful in capping and stabilization of particles. DLS and Zeta potential analysis revealed 2.6 nm average size and −63.9 mV of higher negative zeta potential value indicates greater stability of particles. The crystalline structure and 17.2 nm average size of the particles was revealed by XRD analysis. Microscopic analysis with TEM instrument showed spherical shaped particles with a size range from 5–13 nm. These particles were mostly settled in non-agglomerated and poly-dispersed condition. The software tools like ImageJ and CrystBox supports the calculation of average size and confirms the crystallographic nature of particles. The expository synergistic efficiency of CuO NPs showed growth inhibitory activity of microorganisms as well as MDA-MB-231 breast cancer cell lines. The presence of higher concentration of phenols and proteins in stem bark may be the reason behind for formation of narrow sized particles, bestowed to antimicrobial and anticancer activity. This study concludes that the plant S. alternifolium is an excellent green source for synthesis of copper oxide nanoparticles. This green approach method is very helpful in terms of endemic, endangered medicinal plant i.e., S. alternifolium. Here, the usage of plant material for synthesis is very limited and to produce greater quantities of nanoparticles in a non destructive way. This is the first report on bioinspired green synthesis of CuO NPs from the stem bark extract of S. alternifolium and too proved these nanoparticles as synergistic antimicrobial and anticancer agents. This study may pave a way for the futuristic synthesis of environmentally benign, cost effective CuO NPs from medicinal plants.
The first author is highly grateful to UGC-BSR for providing meritorious fellowship and highly thankful to DST-PURSE, Sri Venkateswara University-Tirupati, SAIF-IIT-Madras, JNTU-Hyderabad for providing characterization facilities. Finally, greatly acknowledged to Prof. K. Suma Kiran, Department of English, Sri Venkateswara University for assisting grammatical and language corrections.
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