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[An Official Publication of ISF College of Pharmacy, Moga]

Original Article
Year : 2020   |  Volume : 12   |  Issue : 2   |  Page : 61-71  

Development and characterization of in situ gel of embelin for the management of Huntington’s disease

Himani Kapahi, Ajay Kumar, Navjot Kaur Sandhu, Puneet Bansal, Neeraj Mishra

Correspondence Address:Department of Pharmaceutics, ISF College of Pharmacy, Moga, Punjab, India, Department of Pharmacology, Government Pharmacy Institute, Patna, Bihar, India, Department of Quality Assurance, ISF College of Pharmacy, Moga, Punjab, India, Department of Pharmacology, Maharaja Ranjit Singh State Technical University, Bathinda, Punjab, India, Amity University, Gwalior, Madhya Pradesh, India

Source of Support: Nil, Conflict of Interest: None Declared

DOI: 10.4103/2231-4040.197331


Aim: This study aims to investigate the in-situ gel for the enhancement of brain drug concentration of embelin for the effective management of Huntington’s disease. Preparation situ gel was prepared by cold method. Optimization: Prepared in situ gel formulation was optimized and characterized by various parameters such as clarity, gelation temperature, gelation time, gel strength, rheological studies, and mucoadhesive force. Gelation time and temperature, mucoadhesive strength of optimized formulation F3 was found to be 30 s, 32–34°C, 1891 dyne/cm2. Optimized formulation was also evaluated by in vitro drug release and in vivo studies such as behavioral parameter, biochemical estimation, and neurological estimations. Results: By these parameters, optimized formulation was found to be suitable for the management of Huntington’s Disease by nasal route because it bypasses the blood-brain barrier by decreasing oxidative stress.

Keywords: Neurodegenerative disorder, in situ gel, Huntington’s disease, nasal delivery, thermoreversible polymers

How to cite this article:
Kapahi H, Kumar A, Sandhu NK, Bansal P, Mishra N. Development and characterization of in situ gel of embelin for the management of Huntington’s disease. Pharmaspire 2020;12(2):61-71.


Neurodegenerative disorders are characterized as progressive loss of function, neuronal dysfunction, and neuronal cell death. The most widely recognized NDs are Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease (HD) and amyotrophic lateral sclerosis, HD.[1] In all cases of degenerative disorders, the aggregation of protein is a distinctive feature.[2]
HD is a fatal, dominantly inherited progressive neurodegenerative disorder, mainly caused by variable-length CAG repeat expansion in the huntingtin (HTT) gene that translates into an abnormally long polyglutamine repeat in the mutant huntingtin protein. The disease is mainly characterized by motor and cognitive impairment, a variable degree of personality change, and psychiatric illness. It also causes progressive disability and death after 15–20 years of onset.[3,4] Accumulation of proteolytic HTT fragments and their aggregation trigger a cascade which leads to an increase in neuronal dysfunction through signals, mitochondrial dysfunction, and energy depletion.[5] These changes are accompanied by neurochemical alterations that involve glutamate receptors and other receptors, such as the dopamine (DA) and adenosine receptors involved in motor functions.[6,7]
Although tetrabenazine has a risk of potentially serious adverse effects, it has DA-depleting potential; hence useful for the treatment of a variety of hyperkinetic movement disorders like neurological diseases characterized as abnormal involuntary movements such as chorea associated with Huntington’s disease.[8] The main goal of the effect of TBZ is to reduce chorea to an acceptable limit to the patient. The goal is not to eliminate chorea completely or to reduce chorea to a level that is acceptable to the caregiver or even physician treating the patient. Some newer antipsychotic agents, such as aripiprazole and olanzapine, may have adequate efficacy with reduced adverse-effect profile than older antipsychotic agents for treating chorea or a neurodegenerative disorder.[9]
Although most gels exhibiting shear-thinning behavior (pseudoplasticity), some gel formulations with suitable rheological properties cannot be easily delivered using a normal nasal spray device. In situ gelation can be used to overcome this problem the advantages of a nasal gel include the reduction of post-nasal drip due to its high viscosity, reduced taste impact due to reduced swallowing, reduction of leakage of the formulation, reduced irritation using soothing excipients, and target delivery to mucosa for better absorption.[10]
Embelia ribes (Family: Myrsinaceae) is a medicinal plant, it was used as an anti-inflammatory agent to relieve rheumatism and fever. Its fruits are used as a brain tonic in treating mental disorders, dyspnoea, heart diseases, etc. Ethanolic extract of E. ribes in streptozotocin-induced diabetes in rats also possesses dyslipidemic and antioxidant activity. Aqueous and ethanolic extracts of embelin were also reported for their potent neuroprotective effects having no major side effects. Embelin (2, 5-dihydroxy-3-undecyl-1,4-benzoquinone) is a naturally occurring alkyl-substituted hydroxy benzoquinone and a major constituent from all the parts of E. ribes. Embelin is reported to possess anti-inflammatory, analgesic, antioxidant, antidiabetic activities, and anticonvulsant potential. Embelin 10 mg/kg and 20 mg/kg dose administration by peroral route were found to be shown good neurodegenerative potential.[11] Embelin from E. ribes has protective effects of on global ischemia/reperfusion-induced brain injury in rats. E. ribes is being used in Indian traditional herbal medicine for the treatment of mental disorders and as a brain tonic.[12] It has been investigated that the pretreatment of embelin (25 and 50 mg/kg, p.o.) significantly increased locomotor activity and hanging latency time and decreased beam walking latency in ischemia disorder.[13]
Embelin exhibited free radical scavenging activities toward diphenyl- 2-picrylhydrazyl (DPPH) radicals with 50% inhibitory concentration. Due to antioxidant and free radical scavenging activity of embelin might be used in Huntington’s disease. Embelin has poor bioavailability approx. 19%. Crude E. ribes displayed free radical scavenging activities when tested using DPPH.[13] The aqueous extract of E. ribes administered orally (100 and 200 mg/kg body weight) showed antioxidant activity against streptozotocin-induced diabetic rats and substantiated the antioxidant activity of embelin.[14,15] Embelin exhibited a natural antioxidant activity against hepatotoxicity induced rats (at a concentration of 20 mg/kg body weight).[16]
In situ gel is a very promising strategy for antioxidant delivery without doing any modification in drug molecule reaches to the brain by olfactory neurons bypassing blood-brain barrier (BBB).[17,18] The olfactory transfer of drugs into the brain is thought to occur by either slow transport inside the olfactory nerve cells to the olfactory bulb or by faster transfer along the perineural space surrounding the olfactory nerve cells into the cerebrospinal fluid surrounding the olfactory bulbs and the brain. Due to their rapid uptake by brain, biodegradable and bioacceptible nature, it is a very effective drug delivery system for brain targeting.[19,20] Furthermore, the practicability in scale-up and lack of burst effect this system is a promising approach to deliver therapeutic entity to the brain in an effective manner.[21] There are so many problems related to nasal delivery such as mucociliary clearance, which facilitates the clearance of nasally applied dosage forms from the nose.[22] Various polymers can be used to achieve more intimate contact with nasal mucosa and result in higher concentration gradient and subsequent increased absorption.[23] In situ gels also offer high drug transport which frequently provided by the longer residence time of the formulation at absorption site like in nasal delivery.[24,25]
In situ, gelation is a process of gel formation at the site of application after the composition or formulation has been applied to the site. It permits the drug to be delivered in a sol form and it converts into gel form at nasal pH (5.5-6.5).[26]
The aim of the present study is nose to brain delivery of embelinloaded in situ gel for the management of Huntington’s disease. The prepared in situ gel was further optimized and characterized for various behavioral, biochemical, and neurochemical parameters. Nasal delivery of in situ gel bypasses BBB and can directly deliver the drug into the brain using nasal olfactory neurons.[27]



Embelin was purchased from Indofine Chemical Company, Inc., USA. Pluronic F-127 (Thermosensitive polymer) was purchased from Sigma-Aldrich Corporation (St. Louis, MO). Carbopol 934 (Gelling agent) was purchased from CDH, New Delhi. All other chemicals and solvents were of analytical reagent grade during the preparation and evaluation of formulation.

Preparation of in situ gel

Mucoadhesive Pluronic-Carbopol gel was prepared by the cold technique prescribed by Schmolka. Carbopol 974 was dissolved in different concentrations (0.1–0.4%) in distilled water and the above mixture was stirred until Carbopol completely dissolves. To the above solution, different concentration of Pluronic F-127 (18–20%) was added for the preparation of in situ gelling liquids. Embelin (2% w/v) was dissolved in methanol and added into the above polymer solution. Benzalkonium chloride (0.01%) was used as a preservative. Partially dissolved pluronic solution was stored in the refrigerator at 4°C overnight and stirred periodically until clear homogenous solutions were obtained and stored for further evaluation parameters.[28]

Characterizations of prepared in situ gel


The clarity of various formulations was determined by visual inspection under black and white background, and it was graded as follows: turbid, +; clear, ++; and very clear (glassy), +++.

pH of formulation

pH of each formulation was determined using pH meter (Equiptronics, Model EQ-610). The pH meter was first calibrated using solutions of pH 4.5 and 7.

Gelation temperature

The temperature at which the liquid phase transforms into the gel is called gelling temperature. To carry out the study, various concentrations of PF-127 and Carbopol 974 were made in distilled water. Gelation temperature was measured by the visual observation method and also using Anton Paar modular compact rheometer MCR52.[29]

Visual observation method

A two-milliliter aliquot of the gel was transferred to a test tube, immersed in a water bath. The temperature of the water bath was increased slowly at a constant rate of 1°C for 2 min from room temperature to the temperature at which the gel formed. The sample was then examined for gelation, which was said to have occurred when the meniscus would no longer move upon tilting the test tube through an angle of 90° using Anton Paar Rheometer (MCR52). The gelation temperature was determined using Anton Paar Rheometer (model: Gmbh, 3ITT) and probe (PP25-SN 17002) using a 1-ml aliquot of the sample. Measurements were performed in oscillation mode using temperature sweep mode. The temperature was increased at a constant rate from 10°C to 60°C. Storage modulus and loss modulus were plotted against temperature automatically using Rheoplus software to determine gelation temperature.[29]

Determination of mucoadhesive strength

Mucoadhesion testing was carried out using a texture analyzer (CT3, Brookfield, USA) with 50 N load cell equipped with a mucoadhesive holder. Goat nasal mucosa was utilized as the model membrane for mucoadhesive strength determination of various formulations. The tissue (about 20 mm×20 mm) was equilibrated for 15 min at 37.0 ± 0.5°C and then placed onto the holder stage of the mucoadhesive holder. The probe was lowered at a rate of 0.5 mm/s until contact with the membrane was made. A contact force of 1 N was maintained for 60 s, and the probe was subsequently withdrawn at a rate of 0.5 mm/s to a distance of 15 mm using the texture analyzer, the maximum force required to separate the probe from the tissue (i.e., maximum detachment force in grams; F) could be detected directly from Texture Pro CT V1.3 Build 14 software.[30]
Mucoadhesive strength = Force of adhesion/Area of force of adhesion
Where force of adhesion 1/4 m (g) (acceleration due to gravity)/1000.

Rheological study

The viscosity of in situ gelling formulations was determined at 25°C with Anton Paar Rheometer (model: Gmbh, 3ITT) and probe (PP25- SN 17002) using a 1-ml aliquot of the sample. Measurements on each value were performed in triplicate at a fixed shear rate of 50/s using Rheoplus software.[28]

Gel strength

A sample of 50 g of the nasal gel was put in a 100-ml graduated cylinder and gelled in a thermostatically controlled water bath at 37°C. A weight of 35 g was placed onto the gel. The gel strength, which is an indication for the viscosity of the nasal gel at physiological temperature, was determined by the time in seconds required by the weight to penetrate 5 cm deep into the gel.[29]

Sol–gel transition temperature measurement

The sol–gel transition temperature (T) of the prepared in situ gel formulations was determined by the water bath method. Brie-y, 2 ml of the prepared formulation was transferred into a test tube (10 ml) with a diameter of 1.0 cm and sealed with a para-lm. The tube was kept in a circulation water bath at 8° sol-gel C and the temperature of the water bath was increased at an increment of 2–3°C in the beginning (from 8°C to 18°C) and then at 0.2–0.5°C until gelation. After each set of the water bath temperature, 10 min was allowed for equilibration. The test tube was then taken out and placed horizontally to observe the state of the sample, and gelation was said to occur when the meniscus would no longer move upon.[31]

In vitro drug release study

In vitro drug release profile of embelin-loaded gel was studied in PBS (pH 5.5) using the dialysis bag method. Dialysis membrane was exposed to running water for 12 h to remove glycerin-based contents. Dialysis membrane was then washed with hot water at 60°C for 3 min and exposed to a 0.3 % (v/v) solution of sulfuric acid for 5 min. This acidification was followed by treatment with hot water to take away excess acid. The treated membrane was reserved in alcohol until used for in vitro drug release studies. In vitro drug release studies were performed using a shaking incubator at a rotation speed of 110 in PBS (pH 5.5) as release medium. Each dialysis bag (pore size: 12 KD, Sigma Chemical Co., St. Louis, MO) was loaded with 2 ml formulation. Volume and temperature of the dissolution medium were 50 ml and 37.0 ± 0.2°C, respectively. At a predetermined time interval, samples (3 ml) were withdrawn, replaced with the same volume of new media, and assayed for drug content at 271 nm against blank using UV–visible spectrophotometer. Mean results of triplicate measurements and standard deviation were reported. Based on various equations, the release kinetic model, followed by the formulations, was calculated.[32]


Age-matched female Wistar rats weighing about (200–250 g) were selected for brain homogenate studies. Animals were obtained from the central animal house facility of Indo Soviet Friendship College of Pharmacy, Moga, Punjab, India. Animals were housed in-group of three in polypropylene cages with husk bedding under standard laboratory conditions of temperature (22 ± 1°C), relative humidity (60%), and light/dark cycle. Rats were fed on standard diet and water libitum. All the behavioral assessments were carried between 9:00 and 17:00 h. The experimental protocol used in this study was approval no. 205 by the Institutional Animal Ethical Committee (IAEC) guidelines for the use and care of experimental animals.

Drug treatment schedule

3-Nitropropionic acid (3-NP) (Sigma Chemicals, St. Louis, MO) was diluted with saline (adjust pH 7.4) and administered intraperitoneally to animals. Drug and in situ gel was administered by the intranasal (i.n.) route through nostrils (50 μl per nostril) by means of Penn Century cannula. All drugs or vehicle were administered daily for 21 days. Animals were randomly divided into different groups, consisting of six animals in each. The study was conducted in multiple phases. On 1st, 7th, 14th, and 21st day, different behavioral parameters were assessed and biochemical and neurochemical parameters were also measured on the 22nd day.
Group I: Control
Group II: 3-Nitropropionic acid
Group III: 3-NP + Embelin (20 mg/kg through intranasal route).
Animal protocol design represents groups of animals and doses of embelin and in situ gel of embelin administered to rats which are shown in Table 1 and experiment protocol design is shown in Figure 1.

In vivo study

In vivo studies were conducted following the protocol by the IAEC. Proper human care of animals was taken during the study period. The in vivo studies were done using Wistar as the animal model includes drug concentration measurement in plasma and organ homogenates in the brain. In vivo studies of optimized formulations were performed by administering the formulations with the help of a suitable delivery device (nasal cannula) to the nasal mucosa from where the formulation reached in brain bypassing the BBB through the olfactory nerve.

Behavioral assessment

Rotarod activity

Rotarod apparatus (INCO Medicraft, Ambala, India) is used for determining the balance, motor coordination, and muscle tone in rats. Motor function and grip strength of the rats were determined using this apparatus. Rats were placed individually on rotating rod and the cutoff time was 300 s and each rat had undergone three separate trials with a gap of 5 min. Briefly, the rats were exposed to the prior training session on the rotarod (constant speed of 25 rpm; rod diameter 7 cm) for 1st, 7th, 10th, and 15th day after 3-NP administration. The average fall of time was recorded and expressed as percentage fall off time.[33]

Measurement of body weight

Animal body weight was recorded on the first and last days of the experimentation. Percent change in body weight was calculated as:
[Body weight (1st day-21st day)×100]/[1st day body weight].

Narrow beam

The apparatus consists of 50 cm wooden strips supported by two pedestals at each end, with a height of 100 cm above the ground. The rats have to traverse a narrow beam which is suspended between a start platform and their home cage. It is important to make sure that the entire apparatus be placed at the height of at least 100 cm above the ground so that the animal fears the height and really attempts to reach the goal box. All rats must be trained to walk over a beam for 4 days before testing. A ceiling of 120 s is employed at the end, after which the rat is removed and placed in the cage by hand and receives if it is the case, a score of 120s. The test procedure is identical for all rats tested and performed in the same environment, preferably in the morning. Time taken to traverse rats from start platform to their home cage was measured along with their number of slipping errors.[33]

Grip strength measurement

Grip strength of the forelimbs was measured using a digital grip force meter (DFIS series, Chatillon, Greensboro, NC, USA). To measure forelimb grip strength, the rat was held by the tail and lowered toward the apparatus. The rat was poisoned the grab the grid with the forelimbs and was gently pulled back to record the grip strength. The grip strength was recorded in kgf.[34]

Dissection and homogenization

On the 22nd day, all the animals were sacrificed by decapitation immediately after behavioral assessments. The brains were removed, forebrain was dissected out, and cerebellum was discarded. Brains were put on the ice, and the cortex and striatum regions were separated and weighed. A 10% (w/v) tissue homogenate was prepared in 0.1 mol/1 phosphate buffer (pH 7.4). Homogenate was centrifuged for 20 min at 20,000 rpm and the supernatant was stored in 80°C for assessing the biochemical parameters.

Biochemical assessment

Biochemical tests were conducted 24 h after the last behavioral test. The animals were sacrificed by decapitation. Brains were removed and rinsed with ice-cold isotonic saline. Brains were then homogenized with ice-cold 0.1 mmol/L phosphate buffer (pH 7.4). The homogenate (10%w/v) was then centrifuged at 10,000 g for 15 min and the supernatant so formed was used for the biochemical estimations.

Estimation of lipid peroxidation (LPO) assay

The extent of LPO in the brain was determined quantitatively by performing the method as described by Wills (1966). The amount of malondialdehyde (MDA) was measured by reaction with thiobarbituric acid at 532 nm using PerkinElmer Lambda 20 spectrophotometer.[35]

Estimation of nitrite

The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide, was determined by a colorimetric assay with Griess reagent (0.1% N-(1-napththyl) ethylenediamine dihydrochloride, 1% sulfanilamide, and 5% phosphoric acid). Equal volumes of the supernatant and the Griess reagent were mixed and the mixture was incubated for 10 min at room temperature in the dark. The absorbance was measured at 540 nm using PerkinElmer Lambda 20 spectrophotometer. The concentration of nitrite in the supernatant was determined from the sodium nitrite standard curve.[36]

Estimation of reduced glutathione (GSH)

Reduced GSH was estimated according to the method described by Ellman. First, 1 ml of supernatant was precipitated with 1 ml of 4% sulfosalicylic acid and cold digested for 1 h at 4°C. The samples were then centrifuged at 1200× g for 15 min at 4°C. To 1 ml of the supernatant obtained, 2.7 ml of phosphate buffer (0.1 mmol/l, pH 8), and 0.2 ml of 5,5`-dithio-bis (2-nitrobenzoic acid) was added. The yellow color developed was measured at 412 nm using PerkinElmer Lambda 20 spectrophotometer.[37]

Neurochemical analysis

Estimation of brain catecholamines

The estimation of brain catecholamines was done by the method described by Patel et al., 2005, with slight modifications. Catecholamines (DA, serotonin, and NE) and their metabolites (3, 4-dihydroxyphenylacetic acid [DOPAC], 5-hydroxyindoleacetic acid [5-HIAA], and homovanillic acid [HVA]) levels in striatum were estimated by HPLC using the electrochemical detector. Waters’ standard system consisting of a high-pressure isocratic pump, a 20 μl manual sample injector valve, C18 reverse phase column, and electrochemical detector was used in the study. The mobile phase consisted of sodium citrate buffer (pH 4.5) – Acetonitrile (87:13, v/v). Sodium citrate buffer consisted of 10 mM citric acid, 25 mM NaH2HPO4, 25 mM EDTA, and 2 mM of 1-heptane sulfonic acid. Electrochemical conditions for the experiment were +0.75 V, sensitivity ranges from 5 to 50 nA. The separation was carried out at a flow rate of 0.8 ml/min. Samples (100 μl) were injected manually. On the day of the experiment, frozen brain samples were thawed and homogenized in a homogenizing solution containing 0.2 M perchloric acid. After that, the samples were centrifuged at 12,000 g for 5 min. The supernatant was filtered through 0.22 mm nylon filters before injecting in the HPLC sample injector. Data were recorded and analyzed with the help of breeze software. Concentrations of neurotransmitters and their metabolites were calculated from the standard curve generated using standard in a concentration range of 10–100 ng/ml. The values are expressed as percentages of the normal control group.[38]

Statistical analysis

All the results are expressed as mean± standard deviation. The treated groups were compared with control by applying the analysis of variance. The statistical analysis was carried out at GraphPad Software Corp. (San Diego, CA). P < 0.05 was considered statistically significant.


Morphology and other parameters

In situ gel of embelin successfully prepared, as shown in Figure 2. During the optimization of ratio of PF-127 and Carbopol 934, 20% (w/v) of PF-127 and 0.3% (w/v) of Carbopol 934 was selected optimum for the incorporation of drugs and with in situ gel. Optimization of PF-127: Carbopol 934 ratio was given below in Table 2. The results of various parameters of optimized formulation were shown in Table 3.


Embelin-loaded in situ gel exhibited rheological behavior at 37°C, as represented in Figure 3. Formulation F3 of in situ gel follows Newtonian flow at below 37°C while at below 37°C, gel shows pseudoplastic behavior. Studies suggested that viscosity decreases with an increase in shear rate, as shown in Figure 3.

Gelling temperature

It is the temperature at which the liquid phase transforms into the gel is determined as the gelling temperature. To carry out the study, various concentrations of PF-127 and Carbopol 934 were made in PBS (pH 5.5), as shown in Table 2. The gelling temperature of formulation F3 was found to be 32–34°C. Finally, various parameters were characterized for formulation F3 like conc. of PF 127 (%) Carbopol 934, gelation temperature, gelation time (s), and mucoadhesive strength (dynes/cm2) [Table 3].

Drug release study

In vitro drug release study

In vitro drug release study from all the formulations showed in Figure 4. In vitro drug release studies of different formulations were performed in PBS pH 5.5 over 150 min. Kinetic analysis of embelin release from saline and embelin from in situ gel indicates that the drug release to be biphasic and the secondary phase followed the Peppas model (which is graphically represented between % releases versus square root of time) with R2 value found to be 0.9983. An in vitro drug release study was of the plain drug in saline shown maximum release, that is, 66.92% and it followed the first-order release with R2 value of 0.9763. The percentage cumulative release of drug from gel-based formulation was comparatively slow than embelin in saline. Further drug-loaded in situ gel has shown a controlled release of the entrapped drug (90%) over the period of 3 h. Studies indicated that Higuchi model of release kinetics, with R2 value of 0.7736, which shows that release occurred through dissolution mechanism. Finally, 33.08% release of embelin-loaded in situ gel was compared with other formulation aimed prolonged release was successfully found. Embelin release from in situ gel followed Higuchi model of release kinetics, as shown in Figure 5 with R2 value of 0.9983 hence shows that the release of drug occurred through diffusion mechanism. Based on the high correlation coefficient, Peppas model was identified as a best-fit model for in situ gel of embelin.
First order release; Y = 0.003x+2.054, R2 = 0.9122
Peppas Model; Y = 1.4223x−1.2736, R2 = 0.9983
Higuchi Model; Y = 4.277x−3.5266, R2 = 0.7336.

In vivo studies

Plasma drug concentration

Embelin (plain) concentration and concentration of in situ gel of embelin in blood plasma were determined at a time interval of 1, 3, 6, 12, and 24 (h). Table 4 showed that the concentration of in situ gel of embelin in blood was higher than that of the concentration of plain embelin. In the case of embelin-loaded in situ gel, 0.070 μg drug reached in systemic circulation after 3 h, but in case of embelin-loaded in situ gel drug reached was 0.035 μg.

Brain drug concentration

Brain drug concentration of embelin was determined at time interval of 1, 3, 6, and 12 h. Figure 6 shows that the concentration of embelin was found to be higher in case of in situ gel as compared to plain embelin because in situ gel system minimize the local and systemic circulation and provide targeted action.

Behavioral assessment

Effect of embelin (in situ gel) on body weight, rotarod activity, grip strength, and narrow beam in rats treated with 3-NP significantly reduced body weight [Figure 7], motor coordination (rotarod) [Figure 8], and increased the latency to reach goal platform (narrow beam test) [Figure 9] (P < 0.05) on 7th, 14th, and 22nd day as compared with the vehicle-treated group, suggesting that the effects of 3-NP most probably mimic either the juvenile onset or late stages of HD-like behavior. The deficiency of GABAergic stimulation has been implicated in the pathophysiology of HD (Lee and Chang, 2004; Saulle et al., 2004). The study suggested the sequential neurodegenerative process in the striatal GABAergic efferent projections during increased neuropathological grades of HD (Allen et al., 2009). In the present study, i.n. administration of in situ significantly improved the behavioral abnormalities in rotarod and locomotor activities (P < 0.05) and decreased the latency to reach the goal platform in narrow beam walk (P < 0.05), indicating the neuroprotective effect. The hunting-like symptoms were reversed at a variable extent on giving treatment using in situ gel as a carrier of two different compositions. Body weight, rotarod activity, grip strength, and narrow beam parameters are shown in Figures 7-11. Body weight, rotarod activity, grip strength, and narrow beam parameter were found to be improved when in situ gel of embelin was given (i.n) to rats pretreated with 3-NP. From these studies, it was observed that in situ gel of embelin showed maximum antioxidant potential in Huntington disease because of its targeted action in brain.
In situ gel and embelin (without carrier) administered through the i.n. route. As the figures of the behavioral activities show that the hunting-like symptoms disappeared on successful treatment with the embelin in forms in situ gel, on comparing the and embelin (without carrier), the in situ gel was found to be effective by the nasal route. The importance of selection of i.n. the route is also observed in the assessment of the biochemical parameter. The 3-NP experiment model mimics both the hyperkinetic and hypokinetic symptoms of HD, depending upon the duration of administration.[39]

Biochemical assessment

Effects of embelin on LPO, nitrite, catalase, and GSH after 3-NP treatment Systemic administration of 3-NP significantly increased LPO, nitrite concentration and GSH in striatum and cortex, and control vehicle-treated found to be stable normal. In situ gel, substantial prove that oxidative damage significantly contributes to the pathogenesis of several neurodegenerative diseases, including HD.[40] Disruption of the mitochondrial enzyme complex activity is associated with ROS. According to the biochemical assays in HD patients show significant increases in MDA and 4-hydroxynonenal brain levels, which are almost 8-fold greater than the control subjects.[41] Both nitrite and nitric oxide species were shown to be oxidizing DA into its quinone forms bound covalently to critical proteins.[42] From the above hypothesis, it can be correlated that 3-NP treatment increased the oxidative stress that is responsible for the damage of dopaminergic neurons (act as motor control), which is ultimately responsible for impairment in motor coordination, behavioral alterations. The values of various parameters of biochemical assessment such as MDA, GSH, and nitrite are shown in Table 5.


Neurochemical estimation

Estimation of brain catecholamines

The estimation of brain catecholamines was done by the method with slight modifications. Catecholamines (DA, serotonin, and norepinephrine [NE]) and their metabolites (DOPAC, 5-HIAA, and HVA) levels in striatum were estimated by HPLC using electrochemical detector. Waters’ standard system consisting of a high-pressure isocratic pump, a 50 μl manual sample injector valve, C18 reverse phase column, and electrochemical detector was used in the study. Mobile phase consisted of sodium citrate buffer (pH 4.5) – Acetonitrile (87:13, v/v). Sodium citrate buffer consisted of 10 mM citric acid, 25 mM NaH2HPO4, 25 mM EDTA, and 2 mM of 1-heptane sulfonic acid. Electrochemical conditions for the experiment were +0.75 V, sensitivity ranges from 5 to 50 nA. Separation was carried out at a flow rate of 0.8 ml/min. Samples (50 μl) were injected manually. On the day of the experiment, frozen brain samples were thawed and homogenized in a homogenizing solution containing 0.2 M perchloric acid. After that, the samples were centrifuged at 12,000 g for 5 min. The supernatant was filtered through 0.22 mm nylon filters before injecting in the HPLC sample injector. Data were recorded and analyzed with the help of breeze software. Concentrations of neurotransmitters and their metabolites were calculated from the standard curve generated using standard in a concentration range of 10–100 ng/ml. The values are expressed as the percentage of the normal control group (Patel et al., 2005). Estimations of various neurochemical parameters such as NE, DA, Serotonin, and their metabolites are shown in Figures 12 and 13, respectively. Figure 12 shows that in situ gel of embelin has higher improvement potential for catecholamines level as compare to plain embelin formulation when compared with a control group of animals whereas, from Figure 13, it was observed that the catecholamine metabolites level in striatum reached to normal (decreased) more progressively and appropriately by in situ gel of embelin when compared with plain embelin.
The method used for the formulation of in situ gel is a cold method. The optimized formulation is F3. The optimized concentration of polymers used for the preparation of in situ gel such as Carbopol 934 and Pluronic F127 are 0.3% and 20%. Formulation F3 of in situ gel shows that follows Newtonian flow can be used for i.n. delivery so that a high amount of drug reaches to brain bypassing BBB. In vitro drug release of embelin follows the Peppas model. % cumulative drug release was also showing the 67% drug release in 150 min at pH 5.5 because the drug which shows the release up to 60% and more lies in the Peppas model. By in vivo study which includes the behavioral, biochemical, and neurochemical parameters if may be concluded that embelin-loaded in situ gel decreases the alteration done by 3-NP, oxidative stress by a significant decrease in the levels of nitrite and MDA or LPO (LPO level) and a significant decrease in the level of GSG. Thus, in situ of embelin is a suitable carrier for nasal delivery and for the management of Huntington’s disease.



This study can be concluded that nose to brain delivery offers the potential advantage for drug delivery to the brain. This research can thus give the right impetus to work in the direction of delivering in situ gel of embelin through the nasal route using. In the present study of embelin-loaded in situ gel administration through the i.n. route shows the beneficial effects due to its antioxidant potential ability to reduce neurodegeneration or both. Thorough behavioral and biochemical parameter, it may clarify the actions of embelin and support the rationale for use in the treatment of motor disorders (oxidative stress). Thus, it can be concluded from the above observations that in situ gel containing embelin found to be a suitable carrier for brain targeting as compared with embelin (without carrier) because it decreases oxidative stress.


We are thankful to Mr. Parveen Garg Honorable Chairman ISF College of Pharmacy, Moga, for providing necessary facilities for the study.

  1. Palmer S, Li J, Wang ZJ, McKeown MJ. Joint amplitude and connectivity compensatory mechanisms in Parkinson’s disease. Neuroscience 2010;166:1110-8.
  2. Cicchetti F, Drouin-Ouellet J, Gross RE. Environmental toxins and Parkinson’s disease: What have we learned from pesticide-induced animal models? Trends Pharmacol Sci 2009;30:475-83.
  3. Nance MA, Myers RH. Juvenile onset Huntington’s disease-clinical and research perspectives. Ment Retard Dev Disabil Res Rev 2001;7:153-7.
  4. Vonsattel JP, DiFiglia M. Huntington disease. J Neuropathol Exp Neurol 1998;57:369-84.
  5. Beal MF, Ferrante RJ. Experimental therapeutics in transgenic mouse models of Huntington’s disease. Nat Rev Neurosci 2004;5:373-84.
  6. Teunissen C, Steinbusch HW, Angevaren M, Appels M, de Bruijn C, Prickaerts J, et al. Behavioural correlates of striatal glial fibrillary acidic protein in the 3-nitropropionic acid rat model: Disturbed walking pattern and spatial orientation. Neuroscience 2001;105:153-67.
  7. Pavese N, Andrews TC, Brooks DJ, Ho AK, Rosser AE, Barker RA, et al. Progressive striatal and cortical dopamine receptor dysfunction in Huntington’s disease: A PET study. Brain 2003;126:1127-35.
  8. Paleacu D. Tetrabenazine in the treatment of Huntington’s disease. Neuropsychiatr Dis Treat 2007;3:545.
  9. Frank S. Tetrabenazine: The first approved drug for the treatment of chorea in US patients with Huntington disease. Neuropsychiatr Dis Treat 2010;6:657-65.
  10. Ghori MU, Mahdi MH, Smith AM, Conway BR. Nasal drug delivery systems: An overview. Am J Pharmacol Sci 2015;3:110-9.
  11. Dhadde SB, Nagakannan V, Roopesh M, Kumar SR, Thippeswamy BS, Veerapur VP, et al. Effect of embelin against 3-nitropropionic acid-induced Huntington’s disease in rats. Biomed Pharmacother 2016;77:52-8.
  12. Thippeswamy B, Nagakannan P, Shivasharan BD, Mahendran S, Veerapur VP, Badami S. Protective effect of embelin from Embelia ribes Burm. Against transient global ischemia-induced brain damage in rats. Neurotox Res 2011;20:379-86.
  13. Mahendran S, Thippeswamy BS, Veerapur VP, Badami S. Anticonvulsant activity of embelin isolated from Embelia ribes. Phytomedicine 2011;18:186-8.
  14. Rao GV, Sateesh K, Mujahidul I, Saber EM. Folk medicines for anticancer therapy-a current status. Cancer Ther 2008;6:913-21.
  15. Surveswaran S, Cai YZ, Corke H, Sun M. Systematic evaluation of natural phenolic antioxidants from 133 Indian medicinal plants. Food Chem 2007;102:938-53.
  16. Bhandari U, Ansari MN. Protective effect of aqueous extract of Embelia ribes Burm fruits in middle cerebral artery occlusion-induced focal cerebral ischemia in rats. Indian J Pharmacol 2008;40:215.
  17. Johal HS, Garg T, Rath G, Goyal AK. Advanced topical drug delivery system for the management of vaginal candidiasis. Drug Deliv 2016;23:550-63.
  18. Joshi R, Kamat J, Mukherjee T. Free radical scavenging reactions and antioxidant activity of embelin: Biochemical and pulse radiolytic studies. Chem Biol Interact 2007;167:125-34.
  19. Goyal G, Garg T, Malik B, Chauhan G, Rath G, Goyal AK. Development and characterization of niosomal gel for topical delivery of benzoyl peroxide. Drug Deliv 2015;22:1027-42.
  20. Goyal G, Garg T, Rath G, Goyal AK. Current nanotechnological strategies for an effective delivery of drugs in treatment of periodontal disease. Crit Rev Ther Drug Carrier Syst 2014;31:89-119.
  21. Garg T, Goyal AK. Biomaterial-based scaffolds-current status and future directions. Expert Opin Drug Deliv 2014;11:767-89.
  22. Marttin E, Merkus FW, Verhoef JC, Schipper NG. Nasal mucociliary clearance as a factor in nasal drug delivery. Adv Drug Deliv Rev 1998;29:13-38.
  23. Critchley H, Davis SS, Farraj NF, Illum L. Nasal absorption of desmopressin in rats and sheep. Effect of a bioadhesive microsphere delivery system. J Pharm Pharmacol 1994;46:651-6.
  24. Garg T, Singh O, Arora S, Murthy R. Scaffold: A novel carrier for cell and drug delivery. Crit Rev Ther Drug Carrier Syst 2012;29:1-63.
  25. Garg T, Singh S, Goyal AK. Stimuli-sensitive hydrogels: An excellent carrier for drug and cell delivery. Crit Rev Ther Drug Carrier Syst 2013;30:369-409.
  26. Bhalerao A, Lonkar SL, Deshkar SS, Shirolkar SV, Deshpande AD. Nasal mucoadhesive in situ gel of ondansetron hydrochloride. Indian J Pharm Sci 2009;71:711.
  27. Dragunow M, Faull RL, Lawlor P, Beilharz EJ, Singleton K, Walker EB, et al. In situ evidence for DNA fragmentation in Huntington’s disease striatum and Alzheimer’s disease temporal lobes. Neuroreport 1995;6:1053-7.
  28. Ibrahim ES, Ismail S, Fetih G, Shaaban O, Hassanein K, Abdellah NH. Development and characterization of thermosensitive pluronic-based metronidazole in situ gelling formulations for vaginal application. Acta Pharm 2012;62:59-70.
  29. Choi HG, Jung JH, Ryu JM, Yoon SJ, Oh YK. Development of in situ-gelling and mucoadhesive acetaminophen liquid suppository. Int J Pharm 1998;165:33-44.
  30. Yong CS, Choi JS, Quan QZ, Rhee JD, Kim CK, Lim SJ, et al. Effect of sodium chloride on the gelation temperature, gel strength and bioadhesive force of poloxamer gels containing diclofenac sodium. Int J Pharm 2001;226:195-205.
  31. Gilbert JC, Washingtona C, Daviesa MC, Hadgraftb J. The effect of solutes and polymers on the gelation properties of pluronic F-127 solutions for controlled drug delivery. J Control Release 1987;5:113-8.
  32. Sharma S, Lohan S, Murthy R. Formulation and characterization of intranasal mucoadhesive nanoparticulates and thermo-reversible gel of levodopa for brain delivery. Drug Dev Ind Pharm 2014;40:869-78.
  33. Kalonia H, Kumar P, Kumar A, Nehru B. Effect of caffeic acid and rofecoxib and their combination against intrastriatal quinolinic acid induced oxidative damage, mitochondrial and histological alterations in rats. Inflammopharmacology 2009;17:211-9.
  34. Khan A, Jamwal S, Bijjem KR, Prakash A, Kumar P. Neuroprotective effect of hemeoxygenase-1/glycogen synthase kinase-3β modulators in 3-nitropropionic acid-induced neurotoxicity in rats. Neuroscience 2015;287:66-77.
  35. Wills E. Mechanisms of lipid peroxide formation in animal tissues. Biochem J 1966;99:667.
  36. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15 N] nitrate in biological fluids. Anal Biochem 1982;126:131-8.
  37. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70-7.
  38. Patel BA, Arundell M, Parker KH, Yeoman MS, O’Hare D. Simple and rapid determination of serotonin and catecholamines in biological tissue using high-performance liquid chromatography with electrochemical detection. J Chromatogr B Analyt Technol Biomed Life Sci 2005;818:269-76.
  39. Borlongan CV, Koutouzis TK, Sanberg PR. 3-Nitropropionic acid animal model and Huntington’s disease. Neurosci Biobehav Rev 1997;21:289-93.
  40. Shivasharan B, Nagakannan P, Thippeswamy BS, Veerapur VP, Bansal P, Unnikrishnan MK. Protective effect of Calendula officinalis Linn. flowers against 3-nitropropionic acid induced experimental Huntington’s disease in rats. Drug Chem Toxicol 2013;36:466-73.
  41. Martyniuk CJ, Fang B, Koomen JM, Gavin T, Zhang L, Barber DS, et al. Molecular mechanism of glyceraldehyde-3-phosphate dehydrogenase inactivation by α, β-unsaturated carbonyl derivatives. Chem Res Toxicol 2011;24:2302-11.
  42. LaVoie MJ, Hastings TG. Dopamine quinone formation and protein modification associated with the striatal neurotoxicity of methamphetamine: Evidence against a role for extracellular dopamine. J Neurosci 1999;19:1484-91.



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