The To synthesise phytoextract mediated nanoparticles using metal salt

The Objective of the present work is aimed to investigate the potency of Bionanoparticles produced using plant extracts and to investigate their biomedical applications. 
Many medicinally important plants are known for their unique Neuroprotective effect and anticancer properties and Neuroprotective effects. Present study will revolve around locally available plants and estimating the best extract to synthesize Nanoscale particles within a short time period. Hence, it will be a step forward in the area of health- and medical-related research. Although , there is a tremendous use of advanced technology towards the betterment of life, the fact can not be ignored that it also has other side effects of damaging the body cells and thus causing severe challenges to life. Advanced technology some time may prove fatal to the life with increased risk of various kinds of diseases. With a rising number of new cases of all types of cancers worldwide, novel agents that could cure or prevent cancers are still very much needed.
Neurodegenerative diseases are on the rise and we need to focus on its prevention and treatment. These are a heterogeneous group of disorders that are characterized by the progressive degeneration of the structure and function of the CNS or PNS. Common neurodegenerative diseases include Alzheimer’s disease and Parkinson’s disease, hence the plants that are selected are widely having Neuroprotective effect hence by producing effective bionanoparticles we can have a new hope in fighing  this age related diseases.
AIMS & OBJECTIVE

Screening and evaluating the experimental plant system for the synthesis of nanoparticle   
Preparation of phytoextract 
To synthesise phytoextract mediated nanoparticles using metal salt silver nitrate (AgNO3) and biological polymer chitosan.
To characterise synthesized nanoparticles using TEM analysis, X-ray diffraction studies, FTIR and NTA analysis
To perform evaluation studies of synthesis of nanoparticles for neuroprotective effect and anticancer properties on Neural and cancer cell lines.

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MATERIALS & METHODS
In the present study, one metal salt i.e Silver nitrate and one biological polymer chitosan will be used to synthesise nanoscale particles. The nanoparticle will be synthesized in the leaf extract of two medicinal plants I.e Convolvulus pluricaulis and Anacardium occidentale independently. Suitable Cell lines for invitro assays for Neuro protective effect and anticancer properties will be procured from National Centre for Cell Science (NCCS) Pune and ACTRAC Mumbai, Respectively.

Collection of experimental plant material

The experimental plant system will be collected from Goa and maintained in the botanical garden of Institute of Science, Mumbai

Preparation of plant extract

3. Characterisation of Nanoparticles 
The synthesized nanoparticles will be characterized using following techniques 
a. UV – Visible spectrometry 
b. NTA analysis
c. X – Ray diffraction studies
d. Transmission electron microscopic analysis
e. FTIR Analysis
4. Neuro Protective Assays 
It would be interesting to demonstrate the effect of this bio NPs to protect cells against various types of insults associated with many neurodegenerative diseases in vitro. Finally a comparative analysis of Bio nanoparticle produced will be carried out to check the efficiency of each for biomedical application. The anticancer and Neuroprotective effects of the various Biosynthesized NPs will be studied and comparative effect of the various combinations will be discussed.
Following three major methods have been considered to be performed for the evaluation of Neuroprotective effect 

Anticholinesterase assays will be performed using Ellman Method (Ellman et al 1961)
Anti oxidant Assay 

The DPPH radical scavenging capacity of each BioNPs will be determined according  to the method suggested by Brand et al (1995)

Anti – inflammatory Assay

Inhibition of protein denaturation will be evaluated following the method of Mizushima et al (1968) and Sakat et al (2010) with slight modification.
5. Anticancer Assays
The anticancer properties will be evaluated in cancer cell lines using following methods 

Cell viability assay: This assay will be performed using Trypan blue exclusion method, McGahon et al 1995
Apoptosis assay: flow cytometry technique will be used for apoptosis assay given by McGahon et al 1995
SRB Assay: Cell cytotoxicity screening by using  Sulfo rhodamine B Assay As given in  Protocol by Vichai et al 2006 will be performed.

EXPECTED RESULTS / OUTCOMES
The Data from test will be used to determine the potential of the synthesized Bio NPs and their application as therapeutic agent for the treatment of Neuro degenerative diseases and Cancer.  The study will demonstrate a novel eco-friendly method of Nanoparticle synthesis from plant      extract and is an attempt in reducing the cost of their production. The study will provide a strong solution to the global rising problem of Neuro degenerative diseases and Cancer, using plant species that are indigenous to and locally available in our country.
The work is planned such a way that it will allow us to Study the Additive combined properties of Nanoparticles and Robust effect of phytoextract constituents on the Cell lines in Vitro. Nanoparticles will help in delivering phytoconstituents into the cell with much more ease and increase the success rate of delivery of capped phytoconstituents compared to delivering it alone

EXPECTED RESULTS FOR NEUROPROTECTIVE EFFECT
Anticholinesterase assays results will allow to  study possible strategies for enhancing brain cholinergic activity in Alzheimer’s disease , Currently acetylcholinesterase inhibitors are most extensively used for Alzheimer’s disease treatment. Physostigmine and tacrine are the only AchE inhibitors reasonably evaluated in Alzheimer’s disease  patients. Their use is limited by the short half?life, peripheral cholinergic side effects, and dose?dependent hepatotoxicity of tacrine. Hence ,the identification of new molecule(s) with fewer side effects in Alzheimer’s disease patients is the need of the hour, the Bio NPs will surely be a safe and natural alternative to these  synthetic drugs and definitely help overcome the above drawbacks of synthetic drugs due to their enhanced stability and biocompatibility.
Denaturation of proteins is a well-documented cause of inflammation. As part of the investigation on the mechanism of the anti-inflammation activity the ability of BioNPs to reduce protein denaturation is expected mainly due to their unique shape, structure and polyphenol capping. Similarly the free radical scavenging properties of synthesized bioNPs is expected to be more due to the presence of phenolic content in the plant extract.
EXPECTED RESULTS FOR ANTI CANCER EFFECT
Expected result for Anti Cancer effect of bioNPs will be an inhibitory effect on cell growth and control of cell proliferation. Cell viability assay will allow us to study the loss of cell viability which is most offen measured as loss of membrane integrity. Apoptosis assay such as Cell size/ granularity will be assessed by flow cytometry method. Cell cytotoxicity screening by SRB Assay will allow us to demonstrate the increase in the cell mortality in all cell lines markedly due to exposure to our BioNPs. It is expected that BioNPs will efficiently penetrate the cancerous cells and arrest the cell cycle due to potent phytoconstituent’s composition.
Extracts of medicinal plants such as Convolvulus pluricaulis are believed to contain a wide array of polyphenolic compounds which might possess Neuroprotective properties and cancer preventive and/or therapeutic properties which will be confirmed from our study.  The data result from this study, will indicate the potential of Anacardium occidentale leaf extract as Neuroprotective and cancer chemopreventive agents. 
The present work is a small attempt towards justifying the meaningful utility of bio resources of our country and to investigate the mode of synthesis of biodegradable and eco-friendly Nanotechnological product using biotechnological concepts for biomedical applications.            

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