1. INTRODUCTION:

Neurotoxicity is a term used to describe neurophysiological changes caused by toxic agents. Neurotoxicants exposure can result in neurocognitive indications and/or psychiatric disturbances. It is considered as a noteworthy reason for neurodegenerative disorders. The most common toxic agents include heavy metals, organophosphates, bacterial and animal neurotoxins and others. Neurotoxicants increases reactive oxygen species which decreases anti-oxidant enzymes causes increased lipid peroxidation this may destroy cholinergic and dopaminergic neurons in brain leads to neurotoxicity. Antioxidant activity of brain is lower than other tissues. so, neural cells are more susceptible to brain damage than other tissues. Neurotoxicity can be chronic, caused by repeated low-level exposure over long periods of time.

The symptoms of neurotoxicity fall along a continuum that range from temporary, minor, and reversible to chronic, quite serious, and potentially leading to permanent brain or nervous system damage (1).

All neurotoxins are not man-made, few are naturally occurring. Naturally occurring metals like Aluminium, Copper, Manganese, Mercury, Lead, Arsenic, Thallium, Fluoride, Mycotoxins and Biotoxins, Water and air pollutants, Few cosmetics, Recreational drugs, excess glutamate and other man-made chemicals like pesticides, solvents, plastics, paints, adhesives, cleaners etc (1,2). We are living in a sea of neurotoxins, and it’s not apparent to evade them all but decrease the overall load. The total number of neurotoxic “burdens.” Dr. William J. Rea of the Environmental Health Center in Dallas, Texas, recommends “massive avoidance” of neurotoxic pollutants found in air, food, and water as the foremost line of defence beside neurotoxicity (3-5).

Neuroprotectants are used for supportive therapy to guard nerve cells from damage because of excess oxygen and chemical toxins. Different studies recommend that natural products, such as polyphenolic and alkaloid compounds that are isolated from plants possibily delayed the neurodegeneration and improved memory and cognitive function. Antioxidant property in neurotoxicants neutralize free radicals and are efficient in prevention of these disorders (6). Naringenin is a trihydroxy flavanone. It is the major flavanone in grapefruit, orange, tomato and other citrus fruits (7). Naringenin is soluble in ethanol and other organic solvents (8). The half life of Naringenin has been stated as 2.3 h (9). Naringenin is a natural polyphenolic compound which has the maximum potential to scavenge ROS. Therefore, the purpose of this study was to prepare and characterize chitosan nanoparticles (CS-NPs) encapsulated with Naringenin and to determine the effect of naringenin (NARN) released from CS-NPs/NARN for improved treatment of neurotoxicity (10)^.Chitosan (CS) is a naturally occurring polysaccharide acquired by partial N-deacetylation of chitin (11). It is used as a nanocarrier owing to its unique properties such as biodegradability, biocompatibility, hydrophilicity and non-toxicity and is also inexpensive (12-14). Tri polyphosphate (TPP) can be used as a cross linker (15,16). CS-NPs produced by ionic cross linking with TPP showed increased drug loading efficiency and also prolonged drug release period (17,18).

2. MATERIALS AND METHODS:

2.1 Materials:

Naringenin (NARN) and Chitosan (CS) were purchased from Sigma Aldrich, Sodium Tripolyphosphate (TPP) from Otto biochemicals, Acetic acid, Potassium Dihydrogen Phosphate and Disodium Hydrogen Phosphate are purchased from Sisco Research Laboratories, India.

2.2 Preparation of NARN loaded CS-NP_S:

Chitosan nanoparticles prepared by ionoic gelation process. Chitosan solution was prepared in the ratio of (0.1% - 0.5% ) by dissolving in 10ml of 1% v/v acetic acid. TPP solution (0.1%w/v) was prepared by dissolving 100mg of TPP in 100ml of deionized water. Add the TPP (10ml) and Naringenin(10ml) solution concurrently drop by drop to the chitosan solution and kept stirring at 2500rpm for 3hours on mechanical stirrer. Nanosuspension is centrifuged at 15,000rpm for 10 min using high-speed centrifuge. Discard the sediment and the preserve the supernatant (19).

2.3 Preformulation studies:

Preformulation studies are generally provides a tool to select suitable excipients compatible with selected drug and play a key role for development of new formulation. It is the first step in development of a drug substance. thus preformulation studies were performed for the acquired sample of drug for identification and compatibility studies. Organoleptic properties, Solubility studies, Compatability studies includes Fourier transform infrared spectroscopy, Differential scanning calorimetry.

2.31 Solubility studies:

Solubility is an significant consideration in nanoparticle formulations as solubility of the solution is an essential requirement. The solubility of drug and polymer was carried out in various solvents such as distilled water, buffer solutions and organic solvents.

2.32 Compatibility studies:

Fourier transform infrared spectroscopy (FTIR):

The fourier transform infrared analysis was conducted for the functional group characterization. FTIR spectra were recorded on PerkinElmer 11 spectrophotometer. Test samples were mixed with KBr, pressed into pellet and scanned from 400 to 4000 cm (20).

Differential Scanning Calorimetry (DSC):

Differential scanning calorimetery (DSC) analysis is used determine drug–excipients compatibility. Here we can see the enthalphy changes. To discover compatibility between drug and excipients a planned temperature rise was applied and resulted thermograms were analyzed by similarity with individual thermograms of components. If no interaction observed then excipients find appropriate to use with selected drug molecule (21).

2.4 Preparation of standard plot of naringenin:

Precisely weighed 100 mg of naringenin dissolved in 100 ml standard volumetric flask using distilled water to get stock solution of 100mg/ml. From this stock solution 10ml of solution was withdrawn and made to 100ml, from there aliquots of 1,2,3,4 and 5ml were withdrawn and further diluted to 10 ml water to obtain a concentration of 10 to 50µg/ml. The absorbance of the solution was measured at 288 nm by using UV spectrophotometer. A graph of concentration Vs absorbance was plotted.

2.5 Characterization of nanoparticles:

Characterization of nanoparticles is based on the size, morphology and surface charge, by means of such advanced microscopic techniques as atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Properties such as the size distribution, average particle diameter, charge influence the physical firmness and the in vivo distribution of the nanoparticles. Properties like surface morphology, size and overall shape are determined by electron microscopy techniques.

Particle size:

Characterizations of nanoparticles are mainly evaluated by the particle size distribution and morphology (22). The fastest and most popular techniques like photon-correlation spectroscopy (PCS) or dynamic light scattering (DLS), broadly used to determine the size of Brownian motion of nanoparticles in colloidal suspensions in the range of nano and submicrons. In this technique solution of spherical particles in Brownian motion causes a Doppler shift when they are exposed against shining monochromatic light (laser). Such monochromatic light exposure hits the moving particle which results in changing the wavelength of the incoming light. Extent of this alteration in wavelength determines the size of the particle (23).

Zeta potential:

Surface charge and intensity determines the interaction of nanoparticles with the biological environment as well as their electrostatic interaction with bioactive compounds. It can be acquired by evaluating the potential difference among the outer Helmholtz plane and the surface of shear. Thus zeta potential of colloidal based dispersion directly assist in evaluating its stability storage. Zeta potential values (high zeta potential values, either positive or negative) are achieved in order to guarantee stability and avoid aggregation of the particles (24).

Surface morphology:

This electron microscopy technique resolves the size, shape and surface morphology with direct visualization of the nanoparticles. During the process of SEM characterization, solution of nanoparticles should be firstly changed into a dry powder. This dry powder is then further mounted on a sample holder followed by coating with a conductive metal (e.g. gold) using a sputter coater. Total sample is then analyzed by scanning with a focused Fi-Ne beam of electrons (25). Average mean size evaluated by SEM is comparable with results obtained by dynamic light scattering. (26).

2.6 Drug content:

The total drug amount in nanosuspension was determined spectrophotometrically. A 0.50-ml aliquot of nanosuspension was evaporated to dryness under reduced pressure at 35 ͦ c. the residue was dissolved in water and filtered with a 0.45µm filter, and naringenin content was assayed spectrophotometrically at 263nm.

2.7 Drug entrapment efficiency:

The entrapment efficiency is also acknowledged as Association Efficiency. The drug-loaded nanoparticles are centrifuged at a high speed of 3500-4000 rpm for 30 min and the supernatant is assayed for non-bound drug concentration by UV spectrophotometer. Entrapment efficiency was calculated as follows:

EE%= Total amount of drug added – Non-bound drug × 100

Total amount of drug added

It was expressed as [%] percentage

2.8 In vitro release studies:

In-vitro diffusion studies (drug release studies) were performed by using diffusion apparatus. A semipermeable membrane was supported on a ring of diffusion cell and 10 ml sample was kept on a membrane in such a way backing layer was phased towards donor compartment. The glass beaker was filled with 100ml of phosphate buffer of (ph:6.8) at a temp 37 ͦ c sample of 2ml was withdrawn at regular intervals from glass beaker for analysis. 2ml of phosphate buffer was replaced instantaneously after sampling to maintain volume equal to 100ml. The absorbance of sampling was measured at 263nm by using uv spectrophotometer. (27-30)

3. RESULTS AND DISCUSSION:

3.1 Compatibility studies:

FTIR:

The FTIR spectroscopic studies were carried out for the Naringenin, Chitosan and a mixture of Naringenin and Chitosan by KBr pellet technique using FTIR spectrophotometer. The spectra of pure NARN showed characteristic bonds in figure 1a,b,c corresponding to C O stretch-ing (1600–1900 cm−1), C O stretching (1603–1632 cm−1), OH-stretching (3100–3290 cm−1), C C stretching (1150 cm−1), C Cstretching (1157–1630 cm−1) and CH, CH2, CH3stretch-ing (2832–3036 cm−1). The CS-NPs/NAR showed OH stretching (3112 cm−1) which was slightly shifted from the previous observation and also showed an increase in energy absorption. The FTIR spectrum of standard is compared with that of mixture and found that there is no interference or no structural changes.

Fig 1: FTIR spectrum of a) Naringenin, b) Chitosan, c) Naringenin and Chitosan

Differential Scanning Calorimetry (DSC):

The DSC studies were carried out for naringenin, Chitosan, Chitosan and naringenin mixture. It was found that there was no interaction between drug and polymer. Thermal analysis indicated that the DSC scan of the drug presented a sharp endothermic peak equivalent to its melting transition temperature shown in figure 2a,b,c. The DSC spectrum of the NARN showed a sharp endothermic peak with heating enthalpy 257.5 J/g.

Fig 2: DSC Spectrum of a) Naringenin, b) Chitosan, c) Naringenin and Chitosan

3.2 Standard plot of Naringenin:

A Standard plot of naringenin was plotted for concentration of 10,20,30,40,50µg/ml with absorbance measured at 288 nm.

Fig 3: Standard graph of Naringenin

Table 1: Standard plot of Naringenin

S. No.	Concentration(µg/ml)	Absorbance
1.	0	0
2.	10	0.096
3.	20	0.188
4.	30	0.269
5.	40	0.347
6.	50	0.426

3.3 Preparation and optimization of Naringenin nanoparticles:

Naringenin loaded chitosan nanoparticles were prepared by ionotropic gelation technique. Other four formulations are prepared with different concentrations of chitosan and optimized. The prepared nanoparticle formulations are found to be turbid and stable and they are packed in airtight containers and stored in a cool place and utilized for further studies.

3.4 Characterization of Nanoparticles:

Measurement of particle size and Zeta potential of Nanoparticles:

Nanoparticle size was determined by means of dynamic light scattering (DLS). All samples were diluted with ultra-purified water and the analysis was accomplished at a scattering angle of 90° and at a temperature of 25^oC.

Table 3: Particle size of Naringenin

S. No.	Formulation	Ratio	Particle Size	Zeta Potential
1.	NC1	1:1	244	43.2
2.	NC2	1:2	233	42.1
3.	NC3	1:3	289	46.4
4.	NC4	1:4	344	49.1
5.	NC5	1:5	486	49.8

From the above table shows that the average particle size was found to be 300 nm

Drug content and entrapment efficiency:

The total drug amount in nanosuspension was determined spectrophotometrically at 290nm (λmax). The EE of NARN-NPs amplified by the way of increasing the concentration of CS as shown in Table I. The EE for NARN-NPs were on the range of 57.5-86.1. Increasing CS concentration from 0.1 % to 0.5 increases EE because the effect of CS-TPP

Table 4: Drug content and entrapment efficiency of Nanoformulation

Formulation	Average Drug Content (mg/ml)	Average Entrapment Efficiency (%)
NC1	0.372	57.5
NC2	0.398	64.6
NC3	0.460	72.8
NC4	0.492	81.4
NC5	0.564	86.1

Scanning Electron Microscope (SEM):

SEM analysis of the prepared formulation was carried out to understand the morphology of Nanoparticles. In the SEM images signifys that the nanoparticles were distinct, uniform and spherical with a smooth surface. thus the images illustrate that appropriate predictable shape has been achieved shown in figure 6.

Fig 4: SEM images of Naringenin and chitosan nanoparticles

In-vitro release studies:

In this NC₁shows the maximum drug release due to low polymer concentration. NC₃, NC₄and NC₅ shows less release of drug owing to higher concentration of polymer. The in-vitro release of NC₂-89.8% illustrated an initial burst release of approximately 60% in the first eight hours, followed by a slow and much reduced additional release for about 24 hours.

Fig 5: In vitro drug release studies of Naringenin

4. CONCLUSION:

Naringenin loaded chitosan nanoparticles are successfully prepared by ionotropic gelation technique for the improvement of neurotoxicity caused by neurotoxicants. Compatibility studies were carried out i.e., DSC, FTIR. There was no incompatibility observed among drug and polymer. Further, characterization studies were performed for all five formulations i.e., particle size, zetapotential, scanning electron microscope (SEM), Drug content, Entrapment efficiency and in-vitro release studies. NC₂has superior particle size than other formulation. The particle size of NC2 was found to be 233nm. Entrapment efficiency of 72.8% and drug release of 89.8%. The CS-NPs/NAR particles were originate to be in spherical shape. Therefore we conclude that Physicochemical properties of CS-NPs/NARN suggested the possibilities of enhancement of penetration across blood brain barrier (BBB) for the improved treatment of neurotoxicity.

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Received on 11.06.2019 Modified on 04.07.2019

Research J. Pharm. and Tech. 2020; 13(1): 129-134.

DOI: 10.5958/0974-360X.2020.00026.8

S. No	INGREDIENTS	NC1	NC2	NC3	NC4	NC5
1.	Naringenin	100mg	100mg	100mg	100mg	100mg
2.	Chitosan	0.1%	0.2%	0.3%	0.4%	0.5%
3.	Tripolyphosphate (0.1%)	10ml	10ml	10ml	10ml	10ml
4.	1% Acetic acid solution	10ml	20ml	30ml	40ml	50ml