Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

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Dr Mohan Z Mani

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On Sep 2018




Prof. Somashekhar Nimbalkar

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Prof. Somashekhar Nimbalkar
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Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
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Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



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Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
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Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
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Dr Saumya Navit
Professor and Head
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Saraswati Dental College
Lucknow
On Sep 2018




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Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




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Best regards,
C.S. Ramesh Babu,
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Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011
Anuradha

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.


Dr. Anuradha
E-mail: anuradha2nittur@gmail.com
On Jan 2020

Important Notice

Original article / research
Year : 2022 | Month : August | Volume : 16 | Issue : 8 | Page : DC22 - DC27 Full Version

In-vitro Antimicrobial Activity of Green Synthesised Silver Nanoparticles of Leaf Extract of Rhinacanthus nasutus against Bacterial Food Borne Pathogens


Published: August 1, 2022 | DOI: https://doi.org/10.7860/JCDR/2022/56581.16713
V Nagasinduja, S Shahitha

1. Research Scholar, Department of Microbiology, Muthayammal College of Arts and Science, Namakkal, Tamil Nadu, India. 2. Assistant Professor, Department of Microbiology, Muthayammal College of Arts and Science, Namakkal, Tamil Nadu, India.

Correspondence Address :
Dr. S Shahitha,
House No. 93C2, Trichy Road, Rasipuram-637408, Namakkal District, Tamil Nadu, India.
E-mail: shahi.aaliya@gmail.com

Abstract

Introduction: Green synthesis of nanoparticles plays a major role in the control of virulent pathogens due to their ecofriendly, depreciated cost and naive nature. This peculiarity of plant based nanoparticle synthesis accomplishes them as fleeting development in nanobiotechnology. Rhinacanthus nasutus is one such plant, which is generally known as snake jasmine has prodigious medicinal properties to explore.

Aim: The present intent of this study was to harmonise silver nanoparticles (AgNPs) from leaves extract of Rhinacanthus nasutus (Snake jasmine) and interrogate its vitality against virulent bacteria secluded from food.

Materials and Methods: The in-vitro study was conducted in Department of Microbiology at Muthayammal College of Arts and Science, Namakkal, Tamil Nadu, India, during November 2018 to November 2020. Nanoparticles were harmonised using 2 mM silver nitrate with leaves extract of Rhinacanthus nasutus which was then characterised by ultraviolet-visible spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM) with Energy Dispersive X-ray (EDX) analysis and Transmission Electron Microscopy (TEM) imaging and Energy Dispersive X-ray (EDX) analysis. Followed with this the antibacterial activity of silver nanoparticles was evaluated by agar well diffusion method against virulent bacteria isolated from food samples. Furthermore AgNPs was subjected to antibiofilm activity. Origin tool and Microsoft word 2010 were used for statistical analysis.

Results: In this present study, 13 food samples were used from which 56 different isolates of various pathogens were isolated. The green synthesis of silver nanoparticles possessed antibacterial activity against antibiotic resistant, biofilm and beta-lactamase producing Escherichia coli, Enterococcus spp, Klebsiella spp, Pseudomonas spp, Proteus spp, Staphylococcus spp, Salmonella spp and Shigella spp. Together with this the amalgamated AgNPs also exhibited antibiofilm activity in all the isolates. The better results of antibiofilm activity was observed against Salmonella species (74.1%).

Conclusion: This study divulges the presence of substantial antibacterial activity of green synthesis of silver nanoparticles of Rhinacanthus nasutus leaves extract against virulent bacterial species. Hence, it can be explored and exploited for the formulation of new antimicrobial against biofilm and betalactamase producing bacterial isolates due to its modest and ecofriendly nature.

Keywords

Antibacterial, Biofilm, Fourier transform infrared spectroscopy, Nanotechnology, Scanning electron microscopy, Transmission electron microscopy

One of the extensively used technologies in modern research is the Nanotechnology. The augmentation of nanoparticles using metals confining biological materials through an ecofriendly modus has fascinated momentous thought. Nanotechnology accords with the molecules size spectrum from 1-100 nm, their amalgamation, and their handling. This science domain uniformly intermingle all the environs of essential science together with biological sciences, chemistry, physics, computational sciences and engineering material sciences for the establishment of nanostructures (1). Discrete methods like biological, chemical and physical methods can be implicated for the amalgamation of nanoparticles.

Nanomaterials have been explored as propitious mechanism for the improvement of diagnostic biosensors, gene and drug delivery and biomedical imaging owing to its nanoscale effects and expanded surface area. Nanotechnology has been proclaimed as the novel industrial revolution, both developing and developed countries are venturing in this technology to reap the market share. The safety and quality of the food can be probably enhanced by the nanotechnology. The detection of pathogens in food systems are improved by the intelligence of nanosensors is been reviewed in few other studies (2),(3). The cell membrane of the pathogenic bacteria can be penetrated by nanoparticles there by interfering with their molecular pathways and formulating exclusive antimicrobial mechanisms. The global crisis or the resistance against the antimicrobials can be limited by the synergy development of nanoparticles and the optimal antibiotics (4). The drug resistance mechanisms in bacteria is deceived by the nanoparticles that may be metallic/carbon/organic nanotubes and also associated with their antimicrobial potential, biofilm formation inhibition or any other important processes (5).

Antibiotic resistant bacteria are evolved by the excessive improper use of antibiotics which finally leads to Multidrug Resistant (MDR) bacteria. Resistance is achieved by a process either by reducing or eliminating the drug effectiveness by the bacteria that undergo such changes in their gene. Such bacteria remain and multiply to cause more severe infection. This resistance in a bacterium is achieved by many mechanisms like mutations, Deoxyribonucleic Acid (DNA) exchange mechanisms and so on (6). Such resistant bacteria cause two fold higher infection risks that may either be clinical or economical finally resulting in delay or even failure in treatment. It is seen highly as severe disease, virulent strain and difficulty in treatment procedures. It may also major cause of high morbidity and mortality in patients (7).

Novel approach that indulges the united use of nanoparticles and plant based antimicrobials to swamp the toxicity concern are also being researched. Blending nature based antimicrobials along with nanoparticles for the inhibition of the activity of efflux pumps in bacteria, quorum sensing intervention and conceivably plasmid curing are few approaches to fight against MDR bacteria. Plants use leaves as their food factories, this is the incentive step for scientists to exploit leaves as nanofactories for manufacturing silver nanoparticles. In spite of easy and uncomplicated design of the leaf extract they have been benefited for the silver nanoparticle production (8).

The ability of antimicrobial activity against fungal and bacterial plant pathogens of the gold and silver nanoparticles produced from a variety of plant extracts were reported in previous research (9). Another interpretation disclose that nanoparticles can infiltrate the bacterial cell membrane and adhere to Nicotinamide Adenine Dinucleotide dehydrogenases (NADH), proliferating a huge amount of Reactive Oxygen Species (ROS), which cause the reduction of Adenosine Triphosphate (ATP) and hinders the respiratory chain. These radicals have the capacity to interact with proteins, sulfur, phosphorus-containing cell constituents, and DNA, destroying those (10).

In another research, it is stated that the ethyl acetate and methanol extracts of Rhinacanthus nasutus leaves were more effective than the standard antibiotics ciprofloxacin and ampicillin discs against Staphylococcus aureus and Klebsiella pneumonia. The silver nanoparticles produced using Rhinacanthus nasutus (Snake jasmine) leaf extract showed the potential antimicrobial activity against both bacteria (Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Escherichia coli) and fungi (Aspergillus niger, Aspergillus flavus) (11).

The silver nanoparticles produced using the methanolic leaf extracts of Rhinacanthus nasutus (Snake jasmine) possessed remarkable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. These silver nanoparticles (AgNps) were highly toxic against different pathogenic bacteria and also to some fungal species there by exhibiting high antibacterial activity and moderate antifungal activity. These nanoparticles are environmentally safe that showed significant medicinal activities hence can be considered or used in medicinal aspects (12). In this current study the antibacterial activity of silver nanoparticles of leaf extracts from Rhinacanthus nasutus were studied against beta-lactamases and biofilm producing bacteria and furthermore antibiofilm activity was also carried out.

Material and Methods

The present in-vitro study was conducted in Department of Microbiology at Muthayammal College of Arts and Science, Namakkal, Tamil Nadu, India, during November 2018 to November 2020. No use of animal models for this work was done, so no ethical clearance certificate was required.

Isolation of Bacteria

Different kinds of fruit juices (apple, orange, pomegranate and papaya) and chicken samples were collected from local shops in Namakkal district. The bacteria were isolated and identified using HiCrome agar (13). The identified bacteria belong to 8 different genera, which were then subjected to antimicrobial study, biofilm production and betalactamase production (14),(15),(16). From these results, highly potential bacteria from each genera were selected for further study.

Plant collection and extract preparation: In the current study, leaves of Rhinacanthus nasutus have been exploited which is possessed from the Salem area, Tamil Nadu, India. The cleaned and air dried leaves were then subjected to grinding for powder formation. A soxhlet extractor was used for the extract preparation using 200 mL of ethanol and acetone as the solvents. The extracts were prepared until a colourless extract was seized from the top of the extractor. Each extract were separately concentrated under reduced pressure. Upon complete evaporation the dry extracts were weighed and used for further studies. Extracts were maintained at a temperature between 2-8oC for further studies (17).

Phytochemical studies: Various phytochemical components i.e, carbohydrates, alkaloids, phenols, quinols, saponins and proteins from the leaves extract of Rhinacanthus nasutus was tested as per the procedure given by Solomon CU et al., procedure (18).

Silver Nanoparticles Preparation

Nanoparticle synthesis with silver nitrate was prepared using the ethanol extract based on the qualitative phytochemical and antibacterial activity to study the qualitative phytochemical and antibacterial activity. Silver nitrate (AgNO3) solution of 2 mM concentration was used, to 5 mL of this solution 0.5 mL of extract was added and observed for the change of dark brown to light brown colour because of the silver ion formation and AgNPs (19). These synthesised nanoparticles were then used for the antibacterial study by agar well diffusion method.

Analysis

Ultraviolet-visible spectrophotometric analysis: The silver nanoparticles synthesised can be better understood by ultraviolet-visible spectrophotometric analysis as it helps to determine the molecular size of the particles. A spectrum scan from 300-800 nm was accomplished out using ultraviolet-visible spectrophotometer to persuade the absorption maxima of synthesised silver nanoparticles (20). The reduction of silver ions and formation of silver nanoparticles developed within an hour of reaction. Control was maintained by using AgNO3.

Fourier Transforms Infrared Spectroscopy (FTI R): A broad range of materials from liquids, pastes, solids, powders, fibres or even from other forms can be investigated either qualitatively or quantitatively by FTIR with a significant standard (21). The synthesised silver nanoparticles solution was centrifuged for 30 minutes at 10000 round per minutes (rpm) to perform FTIR measurements. The free proteins or enzymes were get rid by washing the pellet thrice with 5 mL of deionised water later it is dried in a vacuum drier and then analysed by FTIR.

Scanning electron microscopy (SEM) with Energy dispersive X-ray (EDX) analysis: An electronic beam generates X-rays in this EDX analysis that helps to analyse the quality and quantity of a coating and the underlying surface which can be combined with electron microscopes provides elemental analysis of small areas of even in nanometers (22). The green synthesised AgNPs with ethanol extract of Rhinacanthus nasutus morphology was investigated using SEM coupled with EDX. The AgNPs solution was centrifuged at 10,000 rpm for 20 minutes and drop coated on to thin glass film fabricating and allowing water to completely evaporate and analysed using Zeiss EVO 18 at a voltage of 20kV. The elemental identification and quantitative compositional information were obtained using EDX.

Antibacterial Activity of Rhinacanthus nasutus

Followed by drying of the extract obtained from soxhlet apparatus using vaccum pump, it was then dissolved using ethanol. The antibacterial activity of Rhinacanthus nasutus leaf extract was studied by agar well diffusion method against the isolated 8 bacterial genera (23). In a sterile Muller-Hinton agar plates were taken in which the 24 hours old nutrient broth cultures of test bacteria were swabbed and to which 8 mm well were made using sterile cork borer. Different concentrations of the leaf extract were filled in the wells and were labeled properly along with this reference antibiotic (chloramphenicol, 1 mg/mL of sterile distilled water) and ethanol was also used as control. All the plates were incubated in upright position or 24 hours at 37°C upon incubation the zones were measured. Same procedure was utilised for the synthesised nanoparticles.

Antibiofilm activity of AgNPs: A 96-well μL plate (flat bottom, polystyrene) was used to determine the anti biofilm activity of the AgNPs as described by Mohanta YK et al., (24). The percentage of inhibition of biofilm formation was calculated using following equation:

Percentage biofilm inhibition=[1-(OD620 of cells treated with Ag NPs or plant extracts/OD620 of the non treated control)×100].

Statistical Analysis

Origin tool and Microsoft word 2010 were used for statistical analysis.

Results

In this present study, 13 food samples were used from which 56 different isolates of various pathogens (E. coli-9, Enterococcus spp-9, Klebsiella spp-7, Pseudomonas spp-4, Proteus spp-2, Staphylococcus spp-9, Shigella spp-12 and Salmonella spp-4) were isolated. Out of 56 isolates tested for antibiotic resistance with multiple antibiotics, 32 isolates exhibited resistance above 50% to the tested antibiotics. Out of 56 isolates, 35 isolates were from fruit juice sample and 21 isolates from chicken samples were isolated. Based on the results, it is evident that isolate from fruit juice exhibited more resistance (47.72%) than the isolates from chicken samples (45.89%). Out of this 75% were exhibiting biofilm production property (Table/Fig 1).

By using the soxhlet extraction method, the highest yield was obtained from ethanol extract of 25.4% while the least yield was of acetone (14.5%). Both solvent extracts were subjected to preliminary phytochemical analysis. Alkaloids, flavanoids, phenols, saponins terpenoids and quinols were observed in ethanol extract. In case of acetone extract, following phytochemicals were obtained namely alkaloids, flavanoids, phenols, saponins and terpenoids. The tannins, carbohydrates, quinones and protein were not observed from both solvent extracts. According to phytochemicals results, highest phytochemicals containing ethanol solvent extract was subjected to further analysis (Table/Fig 2).

Presently, both extracts have been subjected for the determination of antibacterial activity against biofilm and betalactamase producing isolates. When ethanol extract was used, Salmonella spp was highly suppressed, revealing a barrier zone of 10-14 mm. The second most suppressed isolate was P. aeruginosa. The 75% of the isolates were suppressed while using 5 mg concentration of extract and 7.5 mg of concentration of extract suppressed all the isolates. While using the control agents of standard antibiotic and ethanol, it did not exhibit any inhibitory activity. The present study revealed that the antibacterial efficacies of ethanol solvent extract of plant was diverse. In this study, when acetone extract was used, the inhibitory zone was obtained from 11 mm to 20 mm; highest inhibitory activity was obtained against to E. coli, Shigella spp and Salmonella spp. Among the eight isolates, E. faecalis and S. aurues were not inhibited by the acetone extracts (Table/Fig 3),(Table/Fig 4). When compared to ethanol extract, acetone solvent extract have been slightly inhibitory activity. Hence, ethanol extract was subjected to silver nanoparticle synthesis.

The green synthesis of silver nanoparticles was successfully carried out using Rhinacanthus nasutus leaf extract (ethanol solvent extract) because the colour of the solution changes from yellowish brown to dark brown, revealing a reduction of silver nitrate in the aqueous solution due to stimulation of surface plasmon vibrations in silver nanoparticles (Table/Fig 5). The ultraviolet-visible absorption spectrum of the synthesised nanoparticles showed a broad peak at 426 nm which is a characteristic band for Ag and this peak indicated that the particles are polydispersed.

The FTIR spectra of R. nasutus leaf extract was shown in (Table/Fig 6). The samples were analysed with plain KBr pellets as blank. The spectral data were compared with a reference to identify the functional groups existing in the sample. The FTIR spectrum of crude plant extract and green synthesised silver nanoparticle incorporated crude extract was compared with reference FTIR spectrum chart to assign a functional group with respect to the obtained peaks (Table/Fig 7),(Table/Fig 8).

The biosynthesised silver nanoparticles were characterised by SEM for their morphology and size. The SEM micrograph showed that the synthesised silver nanoparticles have spherical morphology. The sizes of the nanoparticles were ranged from 18.2-66.1 nm. The EDX result shows the presence of silver ions as the ingredient element (Table/Fig 9). The metallic AgNps generally show a typically strong peak at 3 keV, due to surface plasmon resonance.

In the present investigation, the antibacterial effect of prepared silver nanoparticles was studied on different types of bacteria such as E. coli, K. pneumoniae, P. aeruginosa, Salmonella spp, Proteus spp and Shigella spp (Gram negative) E. faecalis, S. aureus (Gram negative). While using the ethanol extracts of AgNPs, zone of inhibition was ranged from 10-24 mm, among the various bacteria, Salmonella spp was highly suppressed and lowest activity was against S. aureus (Table/Fig 10).

As one of the main findings of this study, AgNPs retard the formation of biofilms of bacterial isolates, which is due to the inhibitory effect of AgNPs on the flagella. In the present study, the antibiofilm activity of AgNPs was evaluated in-vitro against various bacterial isolates; Salmonella spp was treated for 24 hours with AgNPs (5 mg/mL) synthesised from Rhinacanthus nasutus extract and biofilm formation was reduced by 74.1%, same time while using non AgNPs, 44.1% of biofilm formation was reduced.

Discussion

Microbial resistance to antimicrobial agents has become a serious concern worldwide, leading to an increase in mortality in most cases. A research conducted in India implies that there is an upsurge in the resistance and antibiotic use in tertiary hospitals. Therefore, finding new alternatives to the currently used antibiotics has become a necessity. The present study result for isolation of bacteria is correlated with Nagarajan V et al., who observed the presence of various bacterial genera in different food samples (25). Similarly, Babiye B, in his study stated that fresh juices harbour many kinds of enteropathogenic bacteria especially Shigella, Salmonella, Staphylococcus aureus were predominantly seen (26). In a recent study done by Kowalska J et al., they have stated that all the 40 isolates collected from the food samples have formed biofilms (27). Among the 75% of biofilm positive isolates, 83% of isolates were beta-lactamases producing. Sivakumar M et al., revealed the presence of 60.62% beta-lactamase producing bacteria from raw food samples (28).

The results of previous study made on phytochemical components indicate that the ethanolic extract of R. nasutus contains secondary metabolites such as alkaloids, flavonoids, saponins, tannins, steroids, and triterpenes. Likewise the study of Nanthakumar et al., reported that highest inhibitory activity of aqueous extract of Rhinacanthus nasutus than ethanol extract (29). In 2017, Antonysamy J, reported that various solvent extracts of Rhinacanthus nasutus were active against gram positive and gram negative isolates, which is probably attributed to the presence of secondary metabolites (30). In line with previous observations, the present study’s results of phytochemical properties are corroborated with these results also.

The spectral data were compared with a reference to identify the functional groups existing in the sample (31). The metallic AgNps generally show a typically strong peak at 3 keV, due to surface plasmon resonance (32). Similarly, Pasupuleti VR et al., and Giridharan T et al., also characterised the AgNPs of Rhinacanthus nasutus with UV spectra, FTIR, TEM, XRD and DLS analysis (11),(12). Nanoparticles have significantly better performance compared to plant extracts; 5 mg of plant extract does not inhibit bacteria, at the same time killing all bacteria when the sharp sword of nanoparticles rotates (33).

This study was consistent with previous studies, which suggested that the number of phytochemicals containing Rhinacanthus nasutus showed strong antibacterial activity against E. coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Klebsiella pneumonia (11),(26). Several mechanisms have been reported to explain the lethal effect of AgNPs. This might involve the release of Ag+ ions from AgNPs that may attach to the positive charged cell wall, thus leading to the deformation of the proteins and resulting in cell death. On the contrary, some studies suggest that the growth inhibition around the well is due to the release of diffusible inhibitory Reactive Oxygen Species (ROS) from AgNPs. The free radicals from metals may damage the bacterial membranes, mitochondria and DNA, which can subsequently cause bursting and death of the cell. Presently MIC was carryout with ethanol extract of AgNPs. The lowest MIC of 0.5 mg was for Salmonella spp and second most 0.6 mg was for P. aeruginosa.

In order to create biofilms, bacteria must first attach to a surface. Many factors may affect bacterial adherence, including the growing environment and the surface properties of the material. Giridharan T et al., explored the role of AgNPs in bacterial adhesion (12). AgNPs have been shown in several studies to reduce bacterial swarming capability and biofilm formation, which decreases pathogenicity. Plant derived AgNPs have an advantage over non derived AgNPs due to plant metabolite’s ability to act as capping and stabilising agents, improving AgNPs’ antibacterial activity.

Nanoparticles can possess antimicrobial activity depending on their size. Smaller particles are more likely to interact with cells and attach to cytoplasm since less space barriers are present. Additionally, small nanoparticles provide a large area to interact with microorganisms or biological components, making them highly effective. The small size (18.2-66.1 nm) of AgNPs in this study was another contributing factor to its antibacterial and antibiofilm effect. Similarly, Singh P et al., used 15 nm size AgNPs to inhibit the biofilm formation. It is also that reported the antibiofilm potential of AgNPs against human pathogens (34).

In view of the results, it can be concluded that Rhinacanthus nasutus ethanolic leaf extracts of nanoparticles have potential as antimicrobial components against biofilm and betalactamase producing isolates and could be used to treat infectious diseases caused by resistant microbes. Further research is needed to isolate the pure compounds from the leaf extract studied for testing specific activity.

Limitation(s)

In the augmentation of AgNps either by physical or by chemical methods, it comprises of valuable procedure and sometimes the use of chemicals that may cause some ill-effects. Hence, to overcome this, there is a need of clinical trials in animal models and cell cultures which are required to test their antitoxic nature which will be achieved in the future studies.

Conclusion

According to these researchers, nanoparticles with a particle size less than 100 nm have excessive antibiofilm activity. Based on the current findings AgNPs were found to have an effect on biofilm formation. In future these findings can be used in clinical trials in animal models for their efficiency in control of pathogens.

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DOI and Others

DOI: 10.7860/JCDR/2022/56581.16713

Date of Submission: Mar 23, 2022
Date of Peer Review: Apr 30, 2022
Date of Acceptance: May 23, 2022
Date of Publishing: Aug 01, 2022

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? NA
• Was informed consent obtained from the subjects involved in the study? NA
• For any images presented appropriate consent has been obtained from the subjects. NA

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Mar 30, 2022
• Manual Googling: May 19, 2022
• iThenticate Software: Jul 07, 2022 (14%)

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