Antibacterial activity assessment and characterization of green synthesized CuO nano rods using Asparagus racemosus roots extract
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The present investigation aims to synthesize copper oxide nano rods using Asparagus racemosus roots extract (Ar–CuO) in an ecofriendly process. The synthesized CuO nano rods are characterized by XRD, SEM, TEM, FT-IR, Raman and UV–visible spectroscopy. X-ray diffraction pattern showed typical monoclinic structure. SEM and TEM analysis revealed the formation of self-assembled CuO nano rods with diameter and length ~ 50–100 nm and 400–500 nm respectively. The antimicrobial activity of green synthesized copper oxide nanorods was tested against various bacterial pathogens i.e., E. coli, B. subtilus, K. pneumonia, A. hydrophila, P. fluorescens, Y. ruckeri, F. branchiophilum and E. tarda with fascinating action towards harmful bacteria.
KeywordsCopper oxide Nano rods Asparagus racemosus Green synthesis Antibacterial activity MIC Zone of inhibition
Practicing greener pathway in nanotechnology usually inculcates utilization of natural sources, biodegradable and biocompatible materials, non-toxic solvents in a productive and cost effective manner [1, 2, 3, 4]. Green chemistry inspires to design new products and processes that cutback or eliminates the utilization and generation of hazardous substances [5, 6, 7, 8]. Nanotechnology has vast expansion in various fields involving the synthesis of nanoparticles, nanotubes and nanowires due to their surface-enhanced Raman scattering (SERS) and surface plasmon resonance (SPR). Metal oxide nanoparticles have widespread consideration because of their demand in the variety of fields including biomedical sciences, chemical industry, electronics, drug–gene delivery and biosensor etc. . Asparagus racemosus Willd belongs to the family Liliaceae, known as ‘Shatavari’. The major active constituents of Asparagus racemosus roots are steroidal saponins, isoflavones, asparagamine, racemosol, mucilage, polysaccharides, vitamins A, B1, B2, C, E, Mg, P, Ca, Fe, folic acid. It also contains many other chemical constituents that prevents ageing and impart immunity and vitality to the body . It is also used in nervous disorders, dyspepsia, tumors, inflammation, neuropathy and hepatopathy. Its medicinal uses have been reported in the I.P, B.P as well as in Indian traditional systems of medicine (Ayurveda, Unani, and Siddha). Various therapeutic uses have been reported in the literature for this plant. For instance, it is used as anti-ulcer, anti-tussive, anti-neoplastic, anti-stress anti-septic and anti-lithiatic activities. Further, the root extract is also used for immunomodulatory, hypoglycemic, hypolipidemic, antidiarrhoeal, antiseptic, bronchitis, hyperacidity, immunoadjuvant and wound healing activities [11, 12]. Asparagus racemosus Willd is being used in around 64 Ayurvedic formulations that include accustomed formulations such as ‘Shatavarikalpa’, ‘Phalaghrita’ and ‘Vishnu taila’.
Recently, the extract of Aloe Vera plant has been successfully used to synthesize monoclinic phase with average particle size of 20 nm spherical nanoparticles with high yield by the reaction of aqueous metal source ions with the extract of the Aloe Vera plant . Though there are few reports on CuO nano particles using plant extracts [9, 14, 15, 16, 17, 18], to the best of our knowledge, there is no report on synthesis of CuO rod like nanomaterials using A. racemosus root extract. It is well known that nano structured materials have enhanced antibacterial activity due to their high surface area. In the present work, we studied the synthesis of CuO nano rods using A. racemosus root extract for the first time and evaluated the antibacterial activity against various bacterial pathogens. In the present study, we choose fish bacterial pathogens along with human bacterial pathogens as the Aqua culture and fish farming sector growing tremendously from the last decade. However, the major risk in this sector was bacterial infections. In addition, usage of antibiotics is most common in the aquaculture industry, which leads to development of drug resistance along with accumulation of these drugs in fish. Finally, fishes can spread the antibiotics or drug resistance bacteria or infections to human who consumes them, which leads to serious health problems. Therefore, the alternate antibiotic for human as well as fish bacterial pathogens is mandatory.
2 Experimental methods
2.1 Plant material and extraction
Asparagus racemosus plants were collected from Andhra university campus, the roots were separated from the plants, washed under running tap water and shade dried at room temperature. Dried roots were powdered using mixer grinder, mechanically sieved and subjected to soxhlet extraction using deionized water for 24 h. The aqueous solution obtained after extraction is subjected to concentration under reduced pressure by rotary evaporator, after it is dried in hot air oven at 50–60 °C. The crude extract was stored at 4 °C until use.
2.2 Green synthesis of copper oxide nano rods
In order to synthesize CuO nano rods, we have chosen green combustion method with root extract as a fuel. A. racemosus aqueous extract powder (500 mg) was dissolved in 100 mL of distilled water. Copper nitrate (1 M) was first dissolved in 100 mL plant extract solution and poured into RB flask under reflection and stirred at 60 °C for 8 h followed by cooling to room temperature and the supernatant was discarded. The black solid product obtained was centrifuged twice at 3500 rpm for 20 min (using Beckman centrifuge with a Beckman JA-17 rotor), and thoroughly washed to remove all the ions. Further, the synthesized CuO was heated in a Rapid Thermal Annealing (RTA) system at 200 °C for 3 h under nitrogen (N2) and oxygen (O2) flow at ambient atmosphere. The resulting dried precursor was crushed into powder and stored in airtight container for further analysis.
2.3 Characterization of copper oxide nano rods
The optical property of copper oxide nano rods were analyzed with UV–Vis Spectrometer in the range between 240 and 800 nm. The phase identity and purity were identified with X-Ray Diffraction (PANAnalytical—XPERT-PRO). Functional group and chemical composition analysis has been done using FTIR (Shimadzu Prestige 21) spectrometer. Raman spectroscopy was performed using (Horiba HR 8000, Excitation wavelength: 514.5 nm). The shape and morphology of the CuO nano rods were explored by scanning electron microscopy (SEM-Hitachi s-4200N), and energy dispersive X-ray analysis (EDAX) and elemental analysis was done using the same instrument. For further size and crystallinity, TEM, HRTEM images and SAED pattern was done using transmission electron microscope (TEM-1200EX, JEOL Ltd.).
2.4 Antibacterial activity of Ar–CuO nano rods
The antibacterial activity of CuO nano rod like structures was tested against bacterial pathogens using well diffusion method . The test organisms selected include both gram positive bacteria i.e. Bacillus subtilis, Staphylococcus aureus and gram negative bacteria i.e. Escherichia coli, Klebsiella pneumonia, Aeromonas hydrophila, Pseudomonas fluorescence, Flavobacterium branchiophilum, Edwardsiella tarda and Yersinia rukeri. The microbes were cultured in nutrient broth. A 100-Microliter (μL) solution of broth microbial culture was prepared which was then blown out on Muller Hinton agar plates. The plates were left undisturbed for about 10 min to enhance culture absorption. With a sterile gel puncher, the wells (6 mm size) were pierced into the agar. Using a micropipette, a 100-μL solution (50 μg/mL) of CuO nano rods and the positive control gentamycin has been used. The incubation was carried out at a temperature of 37 °C for about 24 h. The size of the inhibition zone was quantified. The same analysis was repeated for three times. The results of the mean and standard error of the mean were presented.
3 Results and discussion
3.1 Characterization of green synthesized copper oxide nano rods
The formation mechanism of green synthesized CuO nano rods without using any additives or surfactants in the presence of aqueous extract of A. racemosus plant solution has been discussed below. The heat release due to addition of plant extract solution can be utilized to enhance the crystal growth. In addition, the presence of phytochemicals with C=O and COO compounds in the plant extract also play a part of role in mechanism. It is speculated that C=O can act as bio-template agent, as well as carboxylate groups favors the directional growth for transformation into CuO nano structures under rapid heat treatment of agglomerated Cu nano rods after centrifugation process . In detail, when copper metal salt was added to plant extract solution, the Cu2+ ions are released from copper nitrate solution due to reduction process using plant extract. The Cu2+ ions react with N–H and –OH compounds, which leads to the formation of metastable state i.e.; Cu(OH)2. Further, the transformation into CuO nano rods from meta stable Cu(OH)2 during biosynthesis process can be explained by considering the role of C=O, and COO groups at 60 °C for 8 h under continuous stirring and by further rapid heat treatment under nitrogen and oxygen gas flow at ambient conditions .
3.2 Antibacterial activity of green synthesized copper oxide nano rod like structures
A facile, unhazardous and green method has been developed to synthesize copper oxide nano rod structures by using root extract of A. racemosus as both reducing and stabilizing agents. The green synthesized CuO nano rods are well characterized using UV–Vis spectra, XRD, SEM, TEM and FTIR analysis. The green synthesized CuO nano rods exhibited potential biological activity against selected human pathogens and fish bacterial pathogens that might be attributed from large surface area of CuO nano rods.
We are thankful to the DST-PURSE Programme (No. SR/PURSE Phase 2/14 dated 22-09-2014) for the financial assistance and Advanced Analytical Laboratory, Andhra University for their support in carrying out in this research work regarding SEM–EDX and XRD, FTIR and UV spectrum analysis.
Compliance with ethical standards
Conflict of interest
The authors declare that there is no conflict of interest.
Statement of human and animal rights
Research does not involving human participants and/or animals.
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