Phytogenic synthesis of silver nanobactericides for anti-biofilm activity against human pathogen H. pylori
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The present study reports the phytogenic synthesis of silver nanobactericides using Acorus calamus L. and their anti-biofilm activity against clinically isolated H. pylori. The synthesis was confirmed with change in the color of the reaction mixture to brown. The increased in the color intensity was periodically monitored with UV–visible spectroscopy which displayed maximum absorption at 410 nm. The biomolecular interaction was studied with FTIR spectral measurements of silver nanobactericides which revealed the presence of broad absorbance band appearing at 3361 is due to OH group and the prominent peak at 1634 correspond to an amide group. X-ray diffraction (XRD) displayed Bragg’s intensities at 2θ angle reflecting (111), (200), (220) and (311) of the face centered cubic (fcc) structure of silver which was compared with standard XRD pattern. The morphological characteristics of nanobactericides were studied using Transmission electron microscopy (TEM) analysis which revealed the polydispersity of nanoparticles with size ranging from 5 to 60 nm. The anti-biofilm activity of silver nanobactericides against H. pylori was measured using crystal violet and ruthenium red assays which revealed 350 µg/mL to be more effective. The obtained activity was validated with standard antibiotics amoxicillin. Overall, the results obtained in the present investigation are promising enough to reveal the efficacy of silver nanoparticles to inhibit the biofilm production.
KeywordsAcorus calamus L. Anti-biofilm H. pylori Crystal violet assay Ruthenium red assay
The emergence and growing rate of drug resistance has resulted in scarcity of potent antibiotics thus affecting all forms of lives [1, 2]. The drug resistant pathogens have high virulence rate which elevate the mortality and morbidity rates . One of the favorable habitats for drug resistant pathogens to explore is human body and hospitals which are often unrecognized . In most of the cases, the normal flora is being treatment without diagnosing the real causative agents. Apart from ESKAPE pathogens which are recognized as the source of drug resistant pathogens, there are untraced pathogenic microbes which are often more dangerous [5, 6]. One such pathogen includes H. pylori which is regarded as one of the most common causes of microbial infections among different communities. According to World health Organization (WHO), H. pylori is ranked as Class I pathogen responsible for various gastric cancers which has been documented with different epidemiological studies which highlights the pathogenicity and its ability to induce cancer. H. pylori is a Gram –ve bacterium which colonizes gastric mucosa and is reported to inhabit and affect nearly half of the world’s population [7, 8]. Morphologically, it is spiral or helical shaped which drives by flagella and moves and disturbs the stomach lining [9, 10]. The disruption caused by H. pylori is also associated with release of gastric acid, gastrin, cytokines and chronic inflammation process . The symptoms may vary from among individual and degree of infection which ranges from nausea, vomiting, loss of appetite, bleeding which may lead to anemia, weakness and fatigue . The recent up gradation of scientific domain with materials functioning at nano-scale has transformed new era of nano-revolution [13, 14]. The progress of nano-related products has influenced all spheres of lives . There are innumerable commercialized products already gained success at global market [14, 16]. There are numerous methodologies to synthesize nanoparticles and most of these methods are not feasible to produce nanoparticles for medical applications . Hence there is new paradigmatic shift towards producing nanoparticles using plants [17, 18, 19, 20]. One such plant includes Acorus calamus L. The selected plant is rhizomatous herb of moist habitats of Asian countries and some parts of America and Europe . Scanty reports on this plant have resulted to have its application in ancient medicine especially in Ayurvedic formulation . To best of our knowledge less explorative studies has been carried on Acorus calamus L. towards evaluating its potential against H. pylori and synthesis of silver nanobactericides. Based on these considerations, the present study was designed and executed towards employing Acorus calamus L. to synthesize silver nanobactericides and their evaluation against clinically isolated H. pylori. Scientific studies report the effect of silver nanobactericides on wide range of microbial pathogens [23, 24, 25, 26]. These properties can be highly valuable against treatment of drug resistant pathogens. To best of our knowledge, scanty reports are available on activity of silver nanoparticles against biofilm of pathogens like H. pylori. Hence the present study reports the brief communication to synthesize silver nanoparticles using Acorus calamus L. and their activity against H. pylori.
2 Materials and methods
2.1 Selection of plant species and preparation of extract
Plant species were selected from abundant growing area of Mysore, Southern India. Initially plants were processed by washing under running tap water followed by double distilled water. Later, plants were treated with 5% of sodium hypochlorite and washed with sterile water and dried aseptically in hot air oven at 50 °C. The dried plant source was powdered in a mixer and was sieved to obtained fine powder which was stored until further experiments.
2.2 Synthesis of nanoparticles from selected plants
The processed plant material was boiled for 30 min to obtain aqueous extract. The aqueous extract obtained was evaluated for synthesis of silver nanoparticles by treating it with 1 mM silver nitrate solution. The synthesis was initially monitored with visual observation followed by analysis under UV–visible spectroscopy in the spectral range of 200–800 nm at different time intervals. The synthesized silver nanoparticles were characterized using FTIR analysis to reveal the phyto-constitutes and functional groups. The crystalline nature of nanoparticles was studied using XRD. The morphological characteristics of nanoparticles were analyzed using TEM [25, 27].
2.3 Anti-biofilm activity of silver nanoparticles
The silver nanoparticles were assessed for anti-biofilm activity against H. pylori as per the protocol described by Filoche et al. (2005) with slight modification. In brief, the cultured biofilm was freshly added with 200 µLHAMF’12 media which was supplemented with tests samples varying different concentration of plant extract and silver nanoparticles ranging from 5,50,200,400,500 µg/mL. The test samples were incubated for 48 and 72 h. The activity of antibiofilm was measured by crystal violet and ruthenium red dye reduction assay .
3 Results and discussion
3.1 Plant preparation for synthesis of nanobactericides
The plant extraction was obtained by heating of plant materials for 3 h on magnetic stirrer. The mixture was sieved using a muslin cloth followed by centrifugation at 8000 rpm for 20 min to remove plant debris. The clear supernatant was collected and aseptically maintained and evaluated for future studies. The aqueous extract was used to synthesize nanobactericides which was initially screened and optimized to achieve the rapid synthesis process by studying the individual parameters like pH, temperature, concentration and ration of metal salts. The initial synthesis was confirmed with change in the color of reaction process from pale yellow to dark brown color. The synthesis of nanoparticles was rapid and completed within 20 min of incubation time. The synthesis was maximum at alkaline pH and elevated temperature above 70 °C. These results are incongruence with previous findings highlighting the worthiness of the parameters [23, 26, 29].
3.2 Characterization of nanobactericides UV–visible spectroscopy analysis
3.3 FTIR analysis of silver nanobactericides
3.4 X-ray diffraction analysis
3.5 TEM, DLS and zeta potential analysis
3.6 Anti-biofilm activity analysis of silver nanobactericides
The results obtained in the present investigation are promising enough to report plant mediated synthesis of nanobactericides and their efficacy for anti-biofilm activity against H. pylori. To best of our knowledge, scanty reports are available on evaluation of nanomaterials for activity against clinically isolated H. pylori. Further study will be valuable to reveal the activity of synthesized silver nanobactericides against different pathogenic bacteria which are significantly resistant to available to standard antibiotics.
Authors acknowledge the Head of the Institute, JSS Academy of Higher Education, Mysuru for the facilities. C. S. greatly acknowledges the funding support from DST DST-SERB (YSS/2015/001135/LS (Ver-I)). Authors are also thankful for the collaborating partners such as Krasnoyarsk State Medical University named after Prof. V. F. Voino-Yasenetskiy and University of Mysore for their participation and timely assistances.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
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