formulation and evaluation of hesperidin-loaded...

279Drug Invention Today | Vol 10 • Issue 3 • 2018

Formulation and evaluation of hesperidin-loaded chitosan nanosuspension for brain targetingI. Somasundaram*, S. Sumathi, S. P. Bhuvaneshwari, K. Mohammed Shafiq, T. S. Shanmugarajan

INTRODUCTIONThe Parkinson’s disease is more prevalent in person with about 65 years of age concomitant with genetic influence, toxins, oxidative stress, mitochondrial abnormalities, free radicals, and alpha-synuclein aggregation in a higher rate, which is considered to be the main cause for Parkinson disease. It is the second most prevalent neurodegenerative extrapyramidal motor disorder succeeding Alzheimer’s disease. Mostly the cause is idiopathic. Mutation in chromosome 4 and 6 is the main origin for Parkinsonism.[1,2] Dopamine is a neurotransmitter which is feasible in performing the functions of locomotion, learning, working, memory, cognition, and emotion. These functions are performed by binding to their specific receptors which are the members of large G-protein-coupled receptor. The

Research Article

Department of Pharmaceutics, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies, Chennai, Tamil Nadu, India

*Corresponding author: I. Somasundaram, Department of Pharmaceutics, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies Pallavaram, Chennai - 600 117, Tamil Nadu, India. Phone:+91-44-22662500/2501/2502/2503. Fax: +91-44-22662513. E-mail: [email protected]

Received on: 25-09-2017; Revised on: 22-10-2017; Accepted on: 28-01-2018

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Website: jprsolutions.info ISSN: 0975-7619

deregulation of the dopaminergic system has a correlation with Parkinson’s disease, generation of pituitary tumors, Tourette’s syndrome, schizophrenia, and attention deficit hyperactive disorder.[3,4]

Oxidation is the main process in augmenting the energy production. However, oxidative stress is the predictable main cause which enhances the progression of disease. This condition arises when there is a reduction in antioxidant defenses or overproduction in reactive oxygen species (ROS). ROS is generated at the time of injury or inflammation. There are several other sites for ROS production such as mitochondrial electron transport, peroxisomal fatty acid, cytochrome p-450, and phagocytic cells. In low concentration, it serves as signaling intermediates for growth, whereas in high concentration, it induces cell injury and death. A free radical is an atom or group with at least one unpaired electron. It is usually an antioxidant molecule that has lost and needs to stabilize itself by stealing an electron from a nearby molecule. Antioxidant can

ABSTRACT

Aim: Parkinson’s disease is characterized by a progressive loss of dopaminergic neuron in the substantia nigra. Hesperidin is a free radical scavenger that has been shown to have a protective effect against oxidative insults. An antioxidant can neutralize the free radical by accepting or donating an electron to eliminate the unpaired condition. Materials and Methods: Hesperidin-loaded chitosan nanosuspension was prepared by ionic gelation method using tripolyphosphate and chitosan. There was no incompatibility observed between the drug and polymer through Fourier-transform infrared and differential scanning calorimetric. Various other parameters such as particle size, zeta potential, scanning electron microscope, drug content, drug entrapment efficiency, and in vitro release have been utilized for the characterization of nanoparticles. Results and Discussion: The average particle size of 188 nm, zeta potential of 48.7 mV, drug content of 0.470 ± 0.25 mg/ml, and entrapment efficiency of 78.2% are obtained with HPN3 formulation. The HPN1 shows the highest drug release followed by HPN2 due to the low concentration of polymer, and HPN4 and HPN5 show less drug release due to the high concentration of polymer. The in vitro release of HPN3 85.2% showed an initial burst release of approximately 60% in the 1st 8 h, followed by a slow and much reduced additional release for about 24 h. Conclusion: HPN3 was chosen as the best formulation due to its reduced particle size and controlled release at optimum polymer concentration which may be taken for the effective treatment of Parkinson’s disease.

KEY WORDS: Chitosan, Hesperidin, Nanoparticles, Parkinson’s disease, Reactive oxygen species

I. Somasundaram, et al.

Drug Invention Today | Vol 10 • Issue 3 • 2018280

neutralize the free radical by accepting or donating an electron to eliminate the unpaired condition.[5-9] Antioxidant is effective in the treatment of cancer, coronary heart disease, and neurodegenerative diseases. It is also used in the treatment of Friedreich ataxia.[10] Bioactive derivatives acting as flavonoids, stilbenoids, and alkaloids possess potent antioxidative and anti-inflammatory properties and are now being inducing in the treatment of Parkinson’s disease.[11] Flavonoids such as hesperidin have been proved to have an antioxidant effect against neurodegenerative disease.[12] Structure-activity relationship of hesperidin to their antioxidant property showed that the effects of antioxidative activity of hesperidin depend on the number and order of OH groups and the presence of C4’-C8’ double bond conjugated pursuing 4-keto group in the flavonoid structure. In addition, it has potential activity in anti-inflammatory, antiviral, anticancer, sedative, Huntington’s stroke, and Alzheimer.

Apparently, the brain needs a regular supply of fuels, gases, and nutrients to maintain homeostasis and other vital function. The blood–brain barrier represents an effective obstacle for the delivery of neuroactive agents to the central nervous system (CNS), and it makes the treatment of many CNS disease difficult to achieve. Activating A2A adenosine receptor (AR) temporarily augments the intracellular spaces between the brain capillary endothelial cells. A2A AR is a member of G protein-coupled receptor. Considering the properties of biodegradable nanoparticles essentially grand bioavailability, control release, less toxicity, and better encapsulation, they are frequently used as a vehicle in drug delivery. Apparently, a used biodegradable nanoparticle includes PLGA, PLA, chitosan, gelatin, polycaprolactone, and poly-alkyl-cyanoacrylates.[13-15] Chitosan is an amino polysaccharide which possesses the idiosyncratic features such as non-toxicity, hydrophilicity, biodegradability, mechanical strength. Biocompatibility and physical inertness are quotidianly used as a carrier for targeted drug delivery in neurodegenerative disorder. It possesses higher penetration capacity, absorption across the mucosal epithelia, and the potential of binding with various ligand molecules and helps in the formation of stable non-complex.[16-21] It is cationic in nature which involves in the formation of stable ionic complexes with multivalent anionic ions or polymers in various forms such as gels and micro/nanoparticles.[22-27] It has an antioxidant activity which is used here as a polymer produced from the exoskeleton of crustaceans (e.g., crabs and shrimps).[28-31] Hence, the present study is carried out to enhance the targeted drug delivery of hesperidin in combination with polymers for the treatment of Parkinson’s disease.

MATERIALS AND METHODSHesperidin, chitosan, and tripolyphosphate (TPP) were purchased from Sigma Aldrich. All the other chemicals used were of analytical grade.

Incompatibility StudiesFourier-transform infrared (FTIR) studyFTIR analysis of pure hesperidin mixture (hesperidin, chitosan, and TPP) was performed, and the spectrum was obtained using FT-IR (Perkin Elmer 200 series). All spectra were recorded within a range of 2000–650 cm−1.

Differential Scanning Calorimetric Analysis (DSC)DSC measurements were carried out using NETZSCH DSC 204. Samples were analyzed in a temperature range of 0°C–300°C at a heating rate of 5°C/min under nitrogen atmosphere. The samples were prepared by pressing them in a DSC aluminum pans and subjected to analysis.

Preparation of Standard Plot of HesperidinAccurately weighed 100 mg of hesperidin dissolved in 100 ml standard volumetric flask using distilled water to get the stock solution of 100 mg/ml. From this stock solution, 10 ml of solution was withdrawn and made to 100 ml, and from these, aliquots of 1, 2, 3, 4, and 5 ml were withdrawn and further diluted to 10 ml with water to obtain a concentration range of 10–50 µg/ml. The absorbance of the solutions was measured at 263 nm using ultraviolet (UV)- spectrophotometer. A graph of concentration versus absorbance was plotted.

Preparation of Hesperidin Nanosuspension and Optimization of Preparation ProcessNanosuspension with relevant stabilizer was prepared by ionotropic gelation method. Chitosan solution (0.1%w/v) was prepared by dissolving chitosan in 100 ml of 1%v/v of acetic acid, and the resulting solution was stirred at 1500 rpm for 30 min on magnetic stirrer (Different concentrations of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% were prepared). TPP solution of 0.1% was prepared by dissolving 100 mg of TPP in 100 ml of deionized water. 100 mg of hesperidin was added to TPP solution and mix to form a homogenous mixture by stirring with a glass rod. Add the above mixture of TPP and hesperidin solution drop by drop (10 ml) to the chitosan solution and kept stirring at 2,500 rpm for 3 h on mechanical stirrer. Nanoparticles were obtained on the addition of a TPP and hesperidin solution to a chitosan solution. The nanoparticle suspension is then centrifuged at 15,000 rpm for 10 min using high-speed centrifuge (Sigma). Discard the sediment and preserve the supernatant. The formation of nanoparticles results in interaction between the negative groups of TPP and the positively charged amino groups of chitosan, as shown in Table 1.



and drop coated on aluminum stub using double-sided carbon tape. The sample was then coated with gold sputter coating unit at 10 Pa vacuum for 10 S (SC7620, Japan). The typical acceleration potential used was 30 kV, and the image was captured at desired magnification.

Drug ContentDrug content was determined by taking 1 ml of the chitosan nanoparticles-loaded hesperidin. To this formulation, 1 ml of aqueous potassium dihydrogen phosphate solution (30 mM) was added and the mixture was centrifuged at 33,000 rpm at 15°C. The clear supernatant was removed and analyzed spectrophotometrically, and the drug content was calculated.

Total drug content = (Volume total/volume aliquot) drug amount in aliquot, where volume total/volume aliquot is a ratio of aliquot of 8th total nanosuspension volume.

Percentage Entrapment EfficiencyThe drug-loaded nanoparticles are centrifuged at 13,000 rpm for 30 min, and the supernatant is assayed for non-bound drug concentration by spectrophotometer.

Entrapment efficiency was calculated as follows:

DEE % = (Drug content/Amount of drug actually present in Nanoparticle suspension)100

In Vitro Release StudiesThe in vitro release profiles of the prepared nanoformulations were studied by diffusion across an artificial membrane. In the donor compartment, nanosuspension containing known concentration of drug was placed and in the receptor compartment buffer was placed and constantly agitated using a magnetic stirrer at 37°C. Samples were withdrawn from the receptor compartment for estimation of released drug and replaced with the same volume of

Table 1: Formulation of hesperidin nanosuspension

Ingredients HPN1 HPN2 HPN3 HPN4 HPN5

Hesperidin (mg)

100 100 100 100 100

Chitosan (%) 0.10 0.20 0.30 0.40 0.500.1% TPP (ml)

100 100 100 100 100

1%acetic acid solution (ml)

100 100 100 100 100

TPP: Tripolyphosphate

Characterization of Hesperidin-Loaded Chitosan NanosuspensionParticle SizeThe size of the prepared nanoparticles was analyzed using Malvern apparatus. All samples were diluted with ultra-purified water, and the analysis was performed at a scattering angle of 90° and at a temperature of 25°C. The mean diameter for each sample and mean hydrodynamic diameter were generated by cumulative analysis in triplicate.

Zeta PotentialThe zeta potential of the prepared nanosuspension was determined using a Malvern zetasizer. The measurements were performed using an aqueous dip cell in an automatic mode by placing diluted samples in the capillary measurement cell and cell position is adjusted. The electrical charge on the nanoparticles was measured by particle electrophoresis (Zetasizer Zen Systems 3600, Malvern Instruments Ltd., UK) after they had been diluted in deionized water to avoid multiple scattering effects and then placed in a folded capillary cell (25°C). Measurements were made in triplicate, and the results are shown as mean ± standard error.

Surface MorphologyScanning electron microscope (SEM)The equipment used was Quanta 200 FEG SEM (FEI Quanta FEG 200). The NPs were dispersed in water

Figure 1: Fourier transform infrared spectrum of hesperidin



buffer. The experiment was carried out in triplicate, and the values were reported as mean value ± standard deviation.

RESULTS AND DISCUSSIONFTIR StudyThe FTIR spectroscopic studies were carried out for the standard hesperidin, chitosan, and a mixture of hesperidin–chitosan by KBr pellet technique using FTIR spectrophotometer. The FTIR spectrum of the standard is compared with that of mixture and found that there is no interference, as shown in Figures 1-3.

DSCThe DSC studies were carried out for hesperidin, chitosan, TPP, and a mixture of hesperidin–chitosan–TPP by DSC. It was found that there is no interaction between drug and polymer, as shown in Figures 4-6.

Standard Plot of HesperidinA standard plot of hesperidin was plotted for the concentration of 10, 20, 30, 40, and 50 µg/ml with the absorbance measured at 263 nm. Calibration equation

was found to be m = y2-y1/x2-x1, as shown in Table 2 and Figure 7.

Characterization of NanoparticlesMeasurement of particle size and zeta potential of nanosuspensionThe particle sizes of prepared nanoparticles were measured from microphotograph of 100 particles. The increase in the concentration of the polymer ratio caused an increase in particle size, as shown in Table 3.

Drug Content and Entrapment EfficiencyHPN3 has more drug content when compared with HPN1 and HPN2. HPN4 and HPN5 have more drug content when compared with HPN3 with more polymer concentration which makes delay in the release of drug, as shown in Table 4.

SEMSEM imaging showed a spherical, smooth, and almost homogenous structure for nanoparticles, as shown in Figures 8 and 9.

Figure 2: Fourier transform infrared spectrum of chitosan

Figure 3: Fourier transform infrared spectrum of mixture of hesperidin and chitosan

Table 2: Standard plot of hesperidin

Concentration (µg/ml) Absorbance (nm)10 0.03820 0.0630 0.08240 0.10450 0.126

Table 3: Particle size and zeta potential of hesperidin nanosuspension

Formulation Ratio Particle Zeta

Size (nm) Potential (mV)HPN1 1:01 148 32.6HPN2 1:02 162 44.8HPN3 1:03 188 48.7HPN4 1:04 227 49.4HPN5 1:05 264 49.7



HPN5 show less drug release due to higher polymer concentration. The in vitro release of HPN3 85.2% showed an initial burst release of approximately 60% in the first eight hours, followed by a slow and much

Table 4: Drug content and entrapment efficiency of hesperidin nanosuspension

Formulation Average drug content (µg/ml)* Average entrapment efficency (%)*HPN1 0.389±0.22 58.9±2.2HPN2 0.435±0.25 65.9±2.5HPN3 0.470±0.25 78.2±2.8HPN4 0.562±0.28 82.1±3.1HPN5 0.611±0.31 85.5±3.1

Table 5: In vitro release study of hesperidin nanosuspension

Time (h)

HPN1* HPN2* HPN3* HPN4* HPN5*

0 0 0 0 0 01 9.8±1.2 8.1±1.3 7.2±1.7 6.7±1.5 6.1±1.32 26.1±1.8 11.2±1.2 10.8±1.5 11.1±2.3 9.2±1.64 48.2±1.4 33.7±1.5 21.2±1.3 22.7±2.6 15.9±2.16 54.1.7 46.1±1.4 33.5±0.9 30±0.9 25.7±1.98 77.4±1.2 57.5±1.3 42.1±2.2 43.1±1.2 36.4±1.812 98.6±1.6 73.1±1.5 54.6±1.9 57.3±1.6 48.3±2.116 - 84.3±1.2 62.3±2.2 63.8±1.1 60.5±1.420 - 97.4±1.7 75.7±1.3 72.2±1.4 68.7±1.324 - - 85.2±1.5 80.2±1.3 75.1±1.7*Values indicates the % cumulative drug release and the results of triplicate trials±S.E.M. SEM: Standard error the mean

Figure 4: Differential scanning calorimetric spectrum of hesperidin

Figure 5: Differential scanning calorimetric spectrum of chitosan

Figure 6: Differential scanning calorimetric spectrum of mixture of hesperidin and chitosan

Figure 7: Standard plot of hesperidin

Figure 8: Scanning electron microscope images of hesperidin in chitosan nanoparticles (HNP3)

In vitro release studiesIn this, HPN1 shows the highest drug release followed by HPN2 due to low polymer concentration. HPN4 and



reduced additional release for about 24 h, as shown in Table 5.

CONCLUSIONThe HPN3 formulation with the hesperidin concentration provided the highest entrapment efficiency of 78.2% when compared to that of other formulations and optimum extent of release (85.2% at 24th h) suggesting the possibility to achieve a therapeutic dose. The particle size was found to be 184 nm and 48.7 mv zeta potential were observed in optimized formulation. Thus, the biodegradable polymers which help in the production of more controlled release dosage form with antioxidant potential of hesperidin may be used for better treatment in Parkinson’s disease.

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Figure 9: Scanning electron microscope images of hesperidin in chitosan nanoparticles (HNP3)

Source of support: Nil; Conflict of interest: None Declared

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