Neuroprotective potential of sesamol and its loaded solid lipid nanoparticles in ICV-STZ-induced cognitive deficits: Behavioral and biochemical evidence

European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Behavioural pharmacology

Neuroprotective potential of sesamol and its loaded solid lipid nanoparticles in ICV-STZ-induced cognitive deficits: Behavioral and biochemical evidence Anand kamal Sachdeva, Shubham Misra, Indu Pal Kaur, Kanwaljit Chopra n University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India

art ic l e i nf o

a b s t r a c t

Article history: Received 6 May 2014 Received in revised form 10 November 2014 Accepted 11 November 2014

Neuroinflammation is a prominent feature of Alzheimer disease (AD) and other chronic neurodegenerative disorders. Intracerebroventricular (ICV) streptozotocin (STZ) induced-cognitive impairment has been widely used as an experimental paradigm of Alzheimer's disease. Sesamol is a potent inhibitor of cytokine production as well as an antioxidant. The present study was designed to evaluate the effectiveness of sesamol in ICV-STZ-induced cognitive deficits in rats by incorporating it into solid lipid nanoparticles (SLNs). ICV-STZ administration produced significant cognitive deficits as assessed by both Morris water maze and elevated plus maze task which is accompanied by significantly enhanced nitrodative stress, altered acetylcholinesterase in rat brain along with significantly increased serum TNFα levels. Chronic treatment with sesamol and sesamol loaded SLNs dose dependently restored cognitive deficits in ICV-STZ rats along with mitigation of nitrodative stress and cytokine release. Effectiveness of SLNs to deliver sesamol to the brain was shown by a significantly better alleviation of the oxidative stress parameters. Our findings demonstrate that loading of sesamol in SLNs in an effective strategy to mitigate ICV-STZ-induced neuronal dysfunction and memory deficits. & 2014 Published by Elsevier B.V.

Keywords: Alzheimer's disease Cognitive deficits Intracerebroventricular Streptozotocin Sesamol Solid lipid nanoparticles

1. Introduction Brain cells are particularly vulnerable to oxidative damage because of their high utilization of oxygen and the substantial polyunsaturated fatty acid content, and this organ has limited ability to combat oxidative stress (Halliwell, 2001; Halliwell and Gutteridge, 1985). Oxidative damage to lipid (lipid peroxidation) and protein (protein carbonyl formation) can lead to structural and functional disruption of the cell membrane, inactivation of enzymes, and finally cell death. Since oxidative damage is implicated in the etiology of neurological complications, treatment with antioxidants has been used as a therapeutic approach in various types of neurodegenerative diseases (Ahmad et al., 2005; Ansari et al., 2004). It has been observed that the use of antioxidants as well as dietary consumption of fruits and vegetables high in antioxidant activity may decrease the risk of memory deficits of the Alzheimer's disease type (Weinstock and Shoham, 2004). Polyphenolic compounds are widely present in plants and they have recently received considerable attention due to their antioxidant n Correspondence to: Pharmacology Research Laboratory, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study, Panjab University, Chandigarh 160014, India. Tel.: þ 91 172 2534105; fax: þ91 172 2541142. E-mail address: [email protected] (K. Chopra).

property. Sesamol (5-hydroxy-1,3-benzodioxole or 3,4-methylenedioxyphenol) is the major constituent of sesame seed oil Sesamum indicum L. (Parihar et al., 2006) and having a powerful antioxidant by inhibits UV- and Fe3 þ /ascorbate-induced lipid peroxidation in rat brain (Prasad et al., 2005; Uchida et al., 1996). Sesamol scavenges hydroxyl and lipid peroxyl radicals and reduces radiation-induced deoxyribose degradation (Joshi et al., 2005). The main issue associated with natural moieties is their poor bioavailability and brain penetration. This issue can be addressed by employing different bioavailability enhancing and brain targeting strategies like solid lipid nanoparticles (SLNs). SLNs are colloidal carriers developed in the last decade as an alternative system to existing traditional carriers (emulsions, liposomes and polymeric nanoparticles). They are a new generation of submicron-sized lipid emulsions where the liquid lipid (oil) has been substituted by a solid lipid. SLNs offer unique properties such as small size, large surface area, high drug loading and the interaction of phases at the interfaces, and are attractive for their potential to improve performance of pharmaceuticals, neutraceuticals and other materials (Cavalli et al., 1993). Sesamol has been shown to possess neuroprotective (Hou et al., 2006; Misra et al., 2011), hepatoprotective (Hsu et al., 2006), antiinflammatory (Chavali et al., 2001; Hou et al., 2006) and anti-ageing properties (Sharma and Kaur, 2006) but still not approved as a therapeutic agent because of its poor bioavailability.

http://dx.doi.org/10.1016/j.ejphar.2014.11.014 0014-2999/& 2014 Published by Elsevier B.V.

Please cite this article as: Sachdeva, A.k., et al., Neuroprotective potential of sesamol and its loaded solid lipid nanoparticles in ICV-STZinduced cognitive deficits: Behavioral and biochemical evidence. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.014i

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SLNs are in the nanometer range, which facilitates their passage across the BBB. However, to bypass reticuloendothelial system (RES) trapping, their particle size should preferentially fall within the range of 120–200 nm. The cells of liver and spleen cannot detect the particles in this range, resulting in an increased blood circulation, thus increasing the time for which the drug remains in contact with the BBB and hence for the drug to be taken up by the brain (Kakkar et al., 2011; Oyewumi et al., 2004). With this background, the present study was designed to investigate the influence of sesamol and sesamol loaded solid lipid nanoparticles (S-SLN) on learning and memory impairments induced by ICV injection of STZ.

2. Materials and methods 2.1. Animals Male wistar rats (200–230 g, 3 months old) bred in the Central Animal House facility of Panjab University, Chandigarh, India were used for the study. The animals had free access to standard rodent food pellets (Ashirwad Industries, Mohali, India) and water. They were acclimatized to the laboratory conditions before the experiment. All the experiments were conducted between 09:00 h and 17:00 h. The experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) of Panjab University, Chandigarh, and performed in accordance with the guidelines of Committee for Control and Supervision of Experimentation on Animals (CPCSEA), Government of India. 2.2. Materials Sesamol and streptozotocin were purchased from Sigma Aldrich, St. Louis, MO, USA. Soy lecithin (Hi Media, India); Tween 80 (S.D. Fine Chemicals Ltd., India), and Compritol 888 ATOs (Glyceryl behenate; gift sample from Gattefosse, USA) were also used in the study. All other chemicals and reagents were of analytical grade and were used without further purification. 2.3. Preparation of sesamol-loaded SLNs Polysorbate 80 (P-80)(45.45%), soy lecithin (0.58%), and water (q.s.) were placed together in a beaker and heated to the lipid melting temperature. Lipid (7.27%) was also melted at 82–85 1C. Sesamol was added to the aqueous phase containing polysorbate 80, following which the hot aqueous emulsifier mix was dropped at once into the lipid melt under magnetic stirring to obtain a clear microemulsion. The hot microemulsion thus formed was transferred slowly into an equivalent amount of cold water (2 1C) under continuous mechanical stirring (280g) for 1.5 h. In the aqueous medium, SLNs are formed by crystallization of the oil droplets present in the microemulsions (Manjunath et al., 2005).The prepared SLNs were stored under refrigeration until further analysis.

Fig. 1. Transmission electron micrograph of sesamol-loaded solid lipid nanoparticles (S-SLNs).

(sesamol being water soluble), and then recentrifuged. The two supernatants were combined, and the absorbance of both the supernatant and the pellet dissolved in methanol: chloroform (1:1) was recorded after appropriate dilutions. The absorbance value obtained for blank SLNs treated in a similar manner was used as the control value to compensate for any interference of the ingredients. All the determinations were performed in triplicate. The amount of drug in the pellet gave a direct measure of the extent of drug entrapped. Entrapment efficiency ¼

Amount of drug=ml of SLN dispersion  Total volume of dispersion  100 Total drug incorporated

Values obtained for the amount of drug in supernatant were used to confirm the mass balance. The particle size and morphology of SLNs was examined using a transmission electron microscope (TEM) (Hitachi H-100; Japan). With sesamol SLNs. TDC was 97.571.63% and Entrapment efficiency was 75.972.91% (n¼6) achieved. The in vitro release was predominantly by diffusion phenomenon and was prolonged up to 7 days (Kakkar et al., 2011). 2.5. Preparation of drug solution/sesamol-SLNs dispersion for dosing Sesamol was dissolved in double distilled water while streptozotocin was dissolved in artificial CSF (ACSF) [(2.9 mM KCl, 147 mM NaCl, 1.7 mM CaCl2, 1.6 mM MgCl2, 2.2 mM D-glucose) in a 25 mg/ml solution]. sesamol-SLNs prepared as above were lyophilized and redispersed in suitable quantities of distilled water (so as to administer 1 mL/dose) for the preparation of different doses ( 4, 8 and 16 mg/kg).

2.4. Characterization of sesamol-loaded SLNs

2.6. Study design:

Developed SLNs (Fig. 1) were spherical when observed under TEM with particle size of around 40–70 nm (Mastersizer 2000, Malvern Instruments, UK). Total drug content (TDC) was estimated spectrophotometrically at λmax of 294 nm by disrupting 1 mL of the prepared SLN dispersion using an appropriate volume of chloroform:methanol (1:1). For determining the entrapment efficiency (EE), the SLN dispersion was ultracentrifuged at 90,558g for 2 h at 481 C, and the clear supernatant was decanted. The pellet of sesamol-loaded (S-SLNs) was then washed with water to remove the unentrapped drug adhering to the surface of nanoparticles

Rats were randomly divided into ten different groups containing 6–8 animals in each group viz Group 1: Control animals received an equivalent volume of vehicle for streptozotocin i.e. artificial CSF (ACSF) on day 1 and day 3; Group 2: animals received intracerebroventricular injection of streptozotocin (ICV-STZ) 3 mg/kg on day 1 and day 3 as a positive control; Group 3, 4 and5: ICV-STZ treated rats being administered sesamol (4, 8, 16 mg/kg respectively) for 21 days; Group 6: ICV-STZ treated rats received unloaded solid lipid nanoparticles (0 mg/kg) for 21 days; Group 7, 8 and 9: ICV-STZ treated rats were administered solid lipid nanoparticles loaded with

Please cite this article as: Sachdeva, A.k., et al., Neuroprotective potential of sesamol and its loaded solid lipid nanoparticles in ICV-STZinduced cognitive deficits: Behavioral and biochemical evidence. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.014i

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sesamol (4, 8 and 16 mg/kg respectively) for 21 days; Group 10: ICVSTZ treated rats were administered Rivastigmine (1.5 mg/kg) as standard for 21 days. Sesamol, sesamol SLNs and rivastigmine treatment was started on day 1, 30 min before the ICV-STZ injection and continued till end of the study i.e. 21 days. Memory impairment was assessed by Morris Water Maze on days 15th to 19th and elevated plus maze on days 20th and 21st. Locomotor activity was measured on day 22nd. After behavioral experiments, rats were anesthetized with thiopentone sodium (40 mg/kg; i.p.), blood was collected through tail vein and animals were killed cervical dislocation. A small cut was made on the neck and further, another small cut was made on scull to isolate the brain. Brain was isolated with the help of microspatula and immediately washed with ice cold normal saline. Brain tissue was stored in  80 1C before preparation of its homogenate and biochemical estimations (Fig. 2).

2.7. Surgical procedures: ICV injection of STZ ICV injection of STZ was made according to the procedure of Sonkusare et al. (2005). Rats were anesthetized with thiopentone (Neon Laboratories, India, 45 mg/kg, i.p.). The scalp was shaved, cleaned and cut to expose the skull. The head was positioned in a stereotaxic frame and a midline sagittal incision was made in the scalp. Burr holes were drilled in the skull on both sides over the lateral ventricles by using the following coordinates: 0.8 mm posterior to bregama; 1.5 mm lateral to sagittal suture and 3.6 mm beneath the surface of the brain (Sharma and Gupta, 2002; Tiwari et al., 2009). Streptozotocin (3 mg/kg, ICV) was injected bilaterally in two divided doses on first and third day making the dose of 1.5 mg/kg each day. The concentration of STZ in ACSF was adjusted so as to deliver 3 μl of the solution. On day one cannula was implanted after streptozotocin administered with the help of dental cement. On day 3rd, the cannula was removed and STZ was administered and wound was sutured. Sham animals received ICV injection of the same volume of ACSF on the first and third day. The skin was sutured after second injection followed by daily application of antiseptic powder (Neosporin). Postoperatively, the rats were fed with oral glucose and normal pellet diet for 4 days, followed by normal pellet diet alone.

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2.8. Behavioral tests 2.8.1. Morris water maze (computer tracking using EthoVision software) Animals were tested in a spatial version of Morris water maze test for assessment of memory (Morris et al., 1982; Tuzcu and Baydas, 2006). The apparatus consisted of a circular water tank (180 cm in diameter and 60 cm high). A platform (12.5 cm in diameter and 38 cm high) invisible to the rats, was set 2 cm below the water level inside the tank with water maintained at 28.5 72 1C at a height of 40 cm. The tank was located in a large room where there were several brightly colored cues external to the maze; these were visible from the pool and could be used by the rats for spatial orientation. The position of the cues remained unchanged throughout the study. The water maze task was carried out for five consecutive days from 15th to 19th day. The rats received four consecutive daily training trials in the following 5 days, with each trial having a ceiling time of 90 s and a trial interval of approximately 30 s. For each trial, each rat was put into the water at one of four starting positions, the sequence of which being selected randomly. During test trials, rats were placed into the tank at the same starting point, with their heads facing the wall. The rat had to swim until it climbed onto the platform submerged underneath the water. After climbing onto the platform, the animal remained there for 20 s before the commencement of the next trial. The escape platform was kept in the same position relative to the distal cues. If the rat failed to reach the escape platform within the maximally allowed time of 90 s, it was guided with the help of a rod and allowed to remain on the platform for 20 s. The time to reach the platform (escape latency in seconds) and total distance traveled to reach the hidden platform (path length in cm) was measured by using computer tracking system with EthoVision software (Noldus Information Technology, Wageningen, Netherlands).

2.8.2. Memory consolidation test A probe trial was performed (Morris et al., 1982; Tuzcu and Baydas, 2006) at the end of 5th day wherein the extent of memory consolidation was assessed. In the probe trial, the rats were placed into the pool for a total duration of 90 s as in the training trial, except that the hidden platform was removed from the pool. Time

Fig. 2. A schematic diagram of drug treatment schedule and protocol design.

Please cite this article as: Sachdeva, A.k., et al., Neuroprotective potential of sesamol and its loaded solid lipid nanoparticles in ICV-STZinduced cognitive deficits: Behavioral and biochemical evidence. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.014i

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spent in target quadrant, was also measured by using computer tracking system with EthoVision software. These parameters indicate the degree of memory consolidation that has taken place after learning. Target quadrant is the quadrant where the platform was previously located before conducting probe trial. 2.8.3. Elevated plus maze test Memory acquisition and retention were tested using elevated plus maze test on days 20 and 21. The apparatus consisted of two crossed arms, one closed and the other, open. Each rat was placed on the open arm, facing outwards. The time taken by the rat to enter the closed arm in the first trial (acquisition trial) on 19th day was noted and was called as initial transfer latency. Cut-off time was fixed at 90 s and in case a rat could not find the closed arm within this period, it was gently pushed in to one of the closed arms and allowed to explore the maze for 30 s. Second trial (retention trial) was performed 24 h after the acquisition trial and retention transfer latency was noted (Sachdeva et al., 2010; Sharma and Gupta, 2002). The retention trial latency was expressed as percentage of initial trial latency. 2.8.4. Closed field activity Spontaneous locomotor activity was measured on day 22 by using computerized actophotometer (IMCORP) for a period of 5 min. Rats were individually placed in a transparent plastic cage (30  23  22 cm3) and were allowed to acclimatize to the observation chamber for a period of 2 min. The locomotion was expressed in terms of total counts per 5 min per animal (Sachdeva et al., 2011). 2.9. Biochemical estimation 2.9.1. Brain homogenate preparation Brain samples were rinsed with ice cold saline (0.9% sodium chloride) and homogenized in chilled phosphate buffer (pH 7.4). The homogenates were centrifuged at 800g for 5 min at 4 1C to separate the nuclear debris. The supernatant thus obtained was centrifuged at 10,500g for 20 min at 4 1C to get the post mitochondrial supernatant, which was used to assay acetylcholinesterase activity, lipid peroxidation, reduced glutathione, nitrite, catalase and superoxide dismutase activity. 2.9.2. Protein estimation The protein was measured by biuret method using bovine serum albumin as standard (Gornall et al., 1949). 2.9.3. Acetylcholinesterase activity Cholinergic dysfunction was assessed by acetylcholinesterase activity. The quantitative measurement of acetylcholinesterase levels in the whole brain homogenate was estimated according to the method of (Ellman et al., 1961). The assay mixture contained 0.05 ml of supernatant, 3 ml of 0.01 M sodium phosphate buffer (pH 8), 0.10 ml of acetylthiocholine iodide and 0.10 ml 5,50 -dithiobis (2-nitro benzoic acid) (Ellman reagent). The change in absorbance was measured at 412 nm for 5 min. Results were calculated using molar extinction coefficient of chromophore (1.36  104 M  1 cm  1) and expressed as percentage of sham group. 2.9.4. Estimation of lipid peroxidation The malondialdehyde content, a measure of lipid peroxidation, was assayed in the form of thiobarbituric acid-reactive substances by the method of Wills (1966). Briefly, 0.5 ml of post-mitochondrial supernatant and 0.5 ml of Tris–HCl were incubated at 37 1C for 2 h. After incubation 1 ml of 10% trichloro acetic acid was added and centrifuged at 1000g for 10 min. To 1 ml of supernatant, 1 ml of 0.67% thiobarbituric acid was added and the tubes were kept in boiling water for 10 min. After cooling, 1 ml double distilled water was

added and absorbance was measured at 532 nm. Thiobarbituric acidreactive substances were quantified using an extinction coefficient of 1.56  105 M  1 cm  1 and expressed as nmol of malondialdehyde per mg protein. Tissue protein was estimated using the Biuret method and the brain malondialdehyde content was expressed as nanomoles of malondialdehyde per milligram of protein. 2.9.5. Estimation of reduced glutathione Reduced glutathione was assayed by the method of Jollow et al. (1974). Briefly, 1.0 ml of post-mitochondrial supernatant (10%) was precipitated with 1.0 ml of sulphosalicylic acid (4%). The samples were kept at 4 1C for at least 1 h and then subjected to centrifugation at 1200g for 15 min at 4 1C. The assay mixture contained 0.1 ml supernatant, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and 0.2 ml 5,50 dithiobis (2-nitro benzoic acid) (Ellman's reagent, 0.1 mM, pH 8.0) in a total volume of 3.0 ml. The yellow color developed was read immediately at 412 nm. 2.9.6. Estimation of superoxide dismutase Cytosolic superoxide dismutase activity was assayed by the method of Kono (1978). The assay system consisted of 0.1 mM EDTA, 50 mM sodium carbonate and 96 mM of nitro blue tetrazolium (NBT). In the cuvette, 2 ml of above mixture was taken and to it 0.05 ml of post mitochondrial supernatant and 0.05 ml of hydroxylamine hydrochloride (adjusted to pH 6.0 with NaOH) were added. The auto-oxidation of hydroxylamine was observed by measuring the change in optical density at 560 nm for 2 min at 30/60 s intervals. 2.9.7. Estimation of catalase Catalase activity was assayed by the method of Claiborne (1985). Briefly, the assay mixture consisted of 1.95 ml phosphate buffer (0.05 M, pH 7.0), 1.0 ml hydrogen peroxide (0.019 M) and 0.05 ml post mitochondrial supernatant (10%) in a final volume of 3.0 ml. Changes in absorbance were recorded at 240 nm. Catalase activity was calculated in terms of K min  1. 2.9.8. Estimation of nitro-dative stress Nitric oxide (nitrate–nitrite) byproducts in brain tissue was determined using the standard total nitric oxide assay kit (Assay Design, Inc. USA). Nitrate was reduced to nitrite by 3 h incubation with nitrate reductase in the presence of nicotinamide adenine dinucleotide 3-phosphate (NADPH). Nitrite was converted to a deep purple azo compound by the addition of Griess reagent. Total nitrite/ nitrate concentration was calculating by using the standard of sodium nitrate. Results were expressed as micromoles/mg protein. 2.9.9. TNF-alpha estimations The quantification of TNF-α was done by the help and instructions provided by R&D Systems Quantikine rat TNF-α immunoassay kit. The Quantikine rat TNF-alpha immunoassay is a 4.5 h solid phase ELISA designed to measure rat TNF-α levels. The assay employs the sandwich enzyme immunoassay technique. A monoclonal antibody specific for rat TNF-α has been pre coated in the microplate. Standards, control and samples were pipette into the wells and any rat TNF-α present is bound by the immobilized antibody. After washing away any unbound substance an enzyme linked polyclonal antibody specific for rat TNF-α is added to the wells. Following a wash to remove any unbound antibody–enzyme reagent, a substrate solution is added to the wells. The enzyme reaction yields a blue product that turns yellow when the stop solution is added. The intensity of the color measured is in proportion to the amount of rat TNF-α bound in the initial steps. The sample values are then read off the standard curve.

Please cite this article as: Sachdeva, A.k., et al., Neuroprotective potential of sesamol and its loaded solid lipid nanoparticles in ICV-STZinduced cognitive deficits: Behavioral and biochemical evidence. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.014i

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2.9.10. Statistical analysis Results were expressed as mean 7S.E.M. The intergroup variation was measured by one-way analysis of variance (ANOVA) followed by Tukey's test using Jandel SigmaStat statistical software version 2.0 (Jandel Corporation, USA). Two-way ANOVA followed by Tukey's test was employed to discover the inter-group variation in escape latency data of the Morris water maze using Jandel Sigma Stat statistical software version 2.0 (Jandel Corporation, USA). A value of P o0.05 was considered statistically significant.

3. Results 3.1. Behavioral observations 3.1.1. Effect of sesamol and sesamol-SLN on performance in Morris water maze task The change in escape latency was observed onto a hidden platform produced by training trials. Although the latencies to reach the submerged platform decreased gradually in all the groups during 5 days of training in Morris water maze test, the mean latency (days 2–5) was significantly (P o0.05) prolonged in ICV-STZ group, as compared to control group, showing a poorer learning performance due to ICV-STZ infusion. This disrupted performance of ICV-STZ group was significantly (P o0.05) improved by the chronic treatment with sesamol (4, 8 and 16 mg/kg) and sesamol-SLN (4, 8 and 16 mg/kg) dose dependent. However, on day 5, sesamol-SLN (8 mg/kg) and sesamol-SLN (16 mg/kg) doses showed a significant difference as compared to sesamol (8 mg/kg) and sesamol (16 mg/kg) respectively. No significant improvement was observed by unloaded SLN as compared to ICV-STZ group. The difference in the mean values among the different levels of Days is greater than would be expected by chance after allowing for effects of differences in treatment group [F(4,49) ¼104.475 (P o0.001)]. The difference in the mean values among the different levels of treatment group is greater than would be expected by chance after allowing for effects of differences in Days[F(9,49) ¼14.615 (Po 0.001)]. The difference in the mean values among the different levels of groups is greater than would be expected by chance after allowing for effects of differences in days. Among the plain sesamol group and sesamol SLNs group, significant difference was observed in 8 mg/kg and 16 mg/ kg dose (Fig. 3A). In the probe trial also, which measures how well the animals had learned and consolidated the platform location during the training, animals showed a significant difference. The time spent in the target quadrant was significantly lower in ICV-STZ rats as compared to the control group. The total time spent in the target quadrant was significantly and dose dependently increased by the chronic sesamol (4, 8 and 16 mg/kg) and sesamol-SLN (4, 8 and 16 mg/kg) treatments, but there was no significant increase in the unloaded SLN (0 mg/kg) treated rats as compared to ICV-STZ treated rats [F(9,49) ¼101.24 (P o0.001)]. Among the plain sesamol group and sesamol SLNs group, significant difference was observed only with 16 mg/kg dose (Fig. 3B). 3.1.2. Effect of sesamol and sesamol-SLN on initial transfer latency in elevated plus maze test Initial transfer latency (ITL) did not differ significantly in any of the groups. Retention transfer latency (RTL) of control group was significantly less than that of ICV-STZ injected group. Treatment with sesamol (4, 8 and 16 mg/kg) and sesamol-SLN (4, 8 and 16 mg/kg) significantly lowered the RTL dose dependently in ICVSTZ injected rats signifying improvement in learning and memory, but there was no significant difference in the RTL between unloaded SLN (0 mg/kg) and ICV-STZ group rats [F(9,49) ¼74.18

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(P o0.001)]. However, sesamol-SLNs (16 mg/kg) was significant decreased RTL as compared to plain sesamol (16 mg/kg) but, there was no significant difference observed between other doses of sesamol (Fig. 3C).

3.1.3. Effect of sesamol and sesamol-SLN on the locomotor activity The spontaneous locomotor activity did not differ significantly between the any groups on the 22nd day [F(9,49) ¼0.897 (P o0.537] (Fig. 4). 3.2. Biochemical observations 3.2.1. Effect of treatment of sesamol and sesamol-SLN on acetylcholinesterase activity Acetylcholinesterase activity was increased in the brains of ICVSTZ treated rats as compared to control group. Sesamol (4, 8 and 16 mg/kg) and sesamol-SLN (4, 8 and 16 mg/kg) significantly and dose-dependently decreased cholinesterase activity in the brain of ICV-STZ injected rats [F(9,49)¼42.26 (Po0.001)]. No significant difference has been observed in the rats treated with unloaded SLN as compared to ICV-STZ group. In between the plain sesamol group and sesamol-SLNs group significant difference was observed between all doses i.e. 4 mg/kg, 8 mg/kg, and 16 mg/kg dose (Table 1).

3.2.2. Effect of treatment of sesamol and sesamol-SLN on antioxidant profile The enzyme activity of catalase, superoxide dismutase and reduced glutathione were significantly decreased in the brain of ICV-STZ treated rats as compared to control group. This reduction was significantly improved by treatment with sesamol (4, 8 and 16 mg/kg) and sesamol-SLN (4, 8 and 16 mg/kg) in ICV-STZ treated rats. Sesamol enhanced the reduced activities of endogeneous antioxidants GSH; there is a statistically significant difference [F(9,49)¼51.37 (Po0.001)], superoxide dismutase; there is a statistically significant difference [F(9,49)¼110.77 (Po0.001)], and catalase; drug treatment, there is a statistically significant difference [F(9,49)¼232.64 (Po0.001)], when administered in ICV-STZ treated rats. However, sesamol plain and sesamol SLNs group, significant difference was observed only with 16 mg/kg dose (Table 1).

3.2.3. Effect of treatment of sesamol and sesamol-SLN on ICV-STZ induced lipid peroxidation Malonaldehyde (MDA) levels were increased significantly in the brain of ICV-STZ treated rats as compared to control group. Chronic treatment with sesamol and sesamol-SLN (4, 8 and 16 mg/ kg) produced a dose dependent and significant reduction in MDA levels in brain of ICV-STZ treated rats [F(9,49) ¼40.11 (P o0.001)]. No significant difference has been observed in the rats treated with unloaded SLN as compared to ICV-STZ group. In between the plain sesamol group and sesamol-SLNs group, significant difference was observed with 16 mg/kg dose only (Table 1).

3.2.4. Effect of treatment of sesamol and sesamol-SLN on ICV-STZinduced nitrosative stress Nitrite levels were significantly elevated in brains of ICV-STZ treated animals as compared to control group. Sesamol and sesamolSLN (4, 8 and 16 mg/kg) treatment significantly (Po0.05) inhibited this increase in nitrite levels in brains of STZ-treated rats [F(9,49)¼ 52.73 (Po0.001)]. No significant difference was observed in the rats treated with unloaded SLN as compared to ICV-STZ group. However, plain sesamol group and sesamol SLNs group, significant difference was observed with 16 mg/kg dose only (Table 1).

Please cite this article as: Sachdeva, A.k., et al., Neuroprotective potential of sesamol and its loaded solid lipid nanoparticles in ICV-STZinduced cognitive deficits: Behavioral and biochemical evidence. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.014i

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Fig. 3. Effect of sesamol (4, 8 and 16 mg/kg) and S-SLN (0, 4, 8 and 16 mg/kg) treatment on the performance of spatial memory acquisition phase using Morris water maze (A) on time spent in target quadrant in probe trial (B) percentage initial transfer latency in elevated plus maze (C) in intracerebroventricular streptozotocin treated rats. Data is expressed as mean7 S.E.M. nPo 0.05, as compared to Control group from second day of the training sessions; #Po 0.05, as compared to ICV-STZ; $,dP o 0.05, as compared to between the treatment group i.e. plain sesamol or sesamol SLN; @Po 0.05, as compared ICV-STZþ sesamol (8 mg/kg); &P o 0.05, as compared to ICV-STZþ sesamol (16 mg/ kg) and ICV-STZþ SLN containing sesamol (16 mg/kg). S(4): Sesamol (4 mg/kg); S(8): Sesamol (8 mg/kg); S(16): sesamol (16 mg/kg); S-SLN (4): sesamol-SLN (4 mg/kg); S-SLN (8): sesamol-SLN (8 mg/kg); S-SLN (16): sesamol-SLN (16 mg/kg); RIVA: Rivastigmine (1.5 mg/kg).

Please cite this article as: Sachdeva, A.k., et al., Neuroprotective potential of sesamol and its loaded solid lipid nanoparticles in ICV-STZinduced cognitive deficits: Behavioral and biochemical evidence. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.014i

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A.k. Sachdeva et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 350

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Fig. 4. Effect of sesamol (4, 8 and 16 mg/kg) and S-SLN (0, 4, 8 and 16 mg/kg) treatment on ambulatory score in actophtometer in intracerebroventricular streptozotocin treated rats. S(4): Sesamol (4 mg/kg); S(8): Sesamol (8 mg/kg); S(16): sesamol (16 mg/kg); S-SLN (4): sesamol-SLN (4 mg/kg); S-SLN (8): sesamol-SLN(8 mg/kg); S-SLN (16): sesamol-SLN (16 mg/kg); RIVA: Rivastigmine (1.5 mg/kg). Table 1 Effect of sesamol (4, 8 and 16 mg/kg) and sesamol-SLN (0, 4, 8 and 16 mg/kg) treatment on the biochemical indices in intracerebroventricular streptozotocin treated rats. Catalase (μM of H2O2/min/mg protein)

Brain nitrite (lg/ml)

AChE (μM/min/mg pr)

3.57 0.02 0.567 0.004a 1.17 0.002b,c

48.90 71.84 5.6772.36a 11.34 71.53b,c

125.837 1.22 215.167 1.52a 188.787 1.15b,c

0.013 70.010 0.077 70.011a 0.065 70.021b,c

0.0127 70.0007b,c

1.6787 0.002b,c

18.9 71.88b,c

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0.0156 70.0018b,c

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0.0069 70.0004a 0.0121 70.0005b,c 0.0156 70.0009b,c 0.0194 70.0007b,c,d 0.0125 70.0016e

7.80 73.18a 10.13 71.34b,c 20.90 71.26b,c 31.46 73.22b,c,d 38.90 71.72e

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0.067 70.013a 0.047 70.012b,c 0.036 70.001b,c 0.021 70.013b,c,d 0.018 70.005e

Groups

LPO (nmoles/mg protein)

GSH (lmoles/mg protein)

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1.017 0.009 2.797 0.01a 2.48 7 0.008b,c

0.0250 70.0005 0.0056 70.0003a 0.0096 70.0002b,c

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SOD (Units/mg protein)

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Data is expressed as mean 7S.E.M. a

p o 0.05, as compared to Control group. p o0.05, as compared to ICV-STZ. c po 0.05, as compared to between the treatment group i.e. plain sesamol or sesamol SLN. d po 0.05, as compared ICV-STZþsesamol (16 mg/kg). e p o 0.05, as compared to ICV-STZþsesamol (16 mg/kg) and ICV-STZþ SLN containing sesamol (16 mg/kg). S-SLN (0): sesamol-SLN (0 mg/kg); S-SLN (4): sesamol-SLN (4 mg/kg); S-SLN (8): sesamol-SLN (8 mg/kg); S-SLN (16): sesamol-SLN (16 mg/kg); RIVA: Rivastigmine (1.5 mg/kg). b

3.2.5. Effect of sesamol and sesamol-SLNs on tumor necrosis factor-alpha (TNF-α) Serum TNF-α level was markedly increased in ICV-STZ group as compared to control. Sesamol and sesamol-SLNs (8 and 16 mg/kg) treatment resulted in significant decrease in serum TNF-α level in ICV-STZ rats as compared to control rats [F(6,34)¼56.46 (Po0.001)]. The marked reduction in TNF-α level at significant extent between plain sesamol and sesamol SLNs was observed in 8 mg/kg and 16 mg/kg dose respectively (Fig. 5).

4. Discussion The ICV-STZ model appears to produce cognitive deficits similar to those seen in Sporadic dementia of Alzheimer's type (Ganguli et al., 2000; Hong and Lee, 1997; Salkovic-Petrisic and Hoyer, 2007). Sporadic dementia is a late onset disease and usually develops after the age of 65. Therefore, adult animals were used in the study to

mimic a situation that more resembles late onset type AD and that is representative of AD dementia. Considering the decreased bioavailability and poor brain penetration of sesamol (Jan et al., 2009), we thought of incorporating sesamol in a colloidal drug carrier system, solid lipid nanoparticles (SLNs). SLNs are taken up readily by the brain because of their lipidic nature, thus are suitable for targeted brain delivery. Decreased escape latency in Morris water maze task in repeated trials demonstrates intact learning and memory function. Sesamol and sesamol-SLN administration decreased the time to reach the hidden platform in Morris water maze task. The results from elevated plus maze further substantiated the findings of Morris water maze test as sesamol and sesamol-SLN reduced the increased percent initial transfer latencies. The locomotor activity did not alter in any treatment group, demonstrating that the latency was not affected by locomotion. There was significant increase in malondialdehyde and nitrite levels along with marked reduction in reduced glutathione and

Please cite this article as: Sachdeva, A.k., et al., Neuroprotective potential of sesamol and its loaded solid lipid nanoparticles in ICV-STZinduced cognitive deficits: Behavioral and biochemical evidence. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.014i

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Fig. 5. Effect of sesamol (4, 8 and 16 mg/kg) and S-SLN (0, 4, 8 and 16 mg/kg) treatment on TNF-α release in ICV-STZ rats. Data is expressed as mean7 S.E.M. nPo 0.05, as compared to Control group; #P o0.05, as compared to ICV-STZ group; $,dPo 0.05, as compared to between the treatment group i.e. plain sesamol or sesamol SLN; @Po 0.05, as compared ICV-STZþ sesamol (8 mg/kg); &P o0.05, as compared ICV-STZþsesamol (16 mg/kg). S(4): Sesamol (4 mg/kg); S(8): Sesamol (8 mg/kg); S(16): sesamol (16 mg/ kg); S-SLN (4): sesamol-SLN (4 mg/kg); S-SLN (8): sesamol-SLN (8 mg/kg); S-SLN (16): sesamol-SLN (16 mg/kg); RIVA: Rivastigmine (1.5 mg/kg).

enzymatic activity of superoxide dismutase and catalase in the brain of ICV-STZ treated rats. Besides the enhanced level of reactive oxygen species, NO levels were also increased in the brains of ICV-STZ treated rats. Peroxynitrite, a harmful oxidant, formed by reaction between superoxide and NO, reacts with a variety of molecules, including protein and non-protein-thiols, unsaturated fatty acids and DNA, thus affecting energy conservation mechanisms and oxidative post-translation modification of protein, and ultimately causing neuronal cell death (Murray et al., 2003). The cognitive restoration by sesamol and sesamol-SLN was coupled with marked inhibition of reactive oxygen and nitrogen species. Our previous laboratory findings have also shown that sesamol ameliorated renoinflammatory cascade and oxidativenitrergic stress in diabetic nephropathy (Kuhad et al., 2009). Various previous studies reported that rivastigmine has also been shown to exert antioxidant activity, possibly through glutameric mechanism (Karam et al., 2014; Yassin et al., 2013). Likewise, rivastigmine treatment significantly attenuated oxidative damage in mice exposed to 3-nitropropionic acid (Klugman et al., 2012; Kumar and Kumar, 2009). Acetylcholine, a neurotransmitter associated with learning and memory, is degraded by the enzyme acetylcholinesterase (AChE), terminating the physiological action of the neurotransmitter. In addition to their role in cholinergic transmission, cholinesterases may also play a role during morphogenesis and neurodegenerative diseases (Reyes et al., 1997). In the present study, the AChE activity was significantly increased in ICV-STZ treated rats as compared to ACSF treated or control rats, which is in accordance with the findings of (Sonkusare et al., 2005). This increase in AChE activity may lead to diminished cholinergic transmission due to a decrease in acetylcholine levels. Sesamol and sesamol-SLN treatment inhibited AChE activity in ICV-STZ treated rats. Sesamol had been shown to inhibit AChE activity in experimental paradigm of diabetes associated cognitive decline (Kuhad and Chopra, 2008). There have been many consistent findings over the years of increased levels of pro-inflammatory cytokines in patients with Alzheimer's disease, e.g. interleukin-1 (IL-1), IL-2, IL-6, IL-8, IL-12, interferon-γ (IFN-γ and tumor necrosis factor-α) (TNF-α) (Schiepers et al., 2005). Systemic exposure to inflammatory challenges, such as lipopolysaccharide (LPS), not only causes a systemic inflammation, but also induces a central neuroinflammation, reflected by activation of brain microglia with a chronically elevated production of

pro-inflammatory mediators. These inflammatory mediators could cause neuronal damage by over-stimulating the immune system, which is supported by the fact that induced brain inflammation in rat causes neurodegeneration and memory loss (Perry et al., 2001). Thus, we examined the involvement of TNF-α in the ICV-STZ induced memory impairment in rats. In the present study, ICV administration of streptozotocin results in elevated TNF-α levels and inflammation. Chronic sesamol and sesamol-SLNs (8 and 16 mg/kg) administration significantly reduces TNF-α levels in ICV-STZ group. Our previous laboratory findings have also shown that sesamol ameliorated inflammatory markers in diabetic neuropathy and diabetes associated cognitive decline (Chopra et al., 2010; Kuhad and Chopra, 2008). In all the above behavioral and biochemical findings it was observed that there was no significant difference between the control group and the SLN (0 mg/kg) (unloaded solid lipid nanoparticle) group, implying that the effect was mainly contributed by sesamol loading as solid lipid nanoparticles. When we compared the plain sesamol and sesamol-SLN group, sesamol-SLN was much more effective than plain sesamol group at 16 mg/kg dose which was almost equivalent to the rivastigmine effects. Therefore, it can be concluded that loading of sesamol in solid lipid nanoparticles has a potential to be explored as a brain targeting strategy for the Alzheimer's disease.

Acknowledgments The Senior Research Fellowship (Anand Kamal Sachdeva) of the Council of Scientific and Industrial Research (CSIR), New Delhi, is gratefully acknowledged. References Ahmad, M., Saleem, S., Ahmad, A.S., Yousuf, S., Ansari, M.A., Khan, M.B., Ishrat, T., Chaturvedi, R.K., Agrawal, A.K., Islam, F., 2005. Ginkgo biloba affords dosedependent protection against 6-hydroxydopamine-induced parkinsonism in rats: neurobehavioural, neurochemical and immunohistochemical evidences. J. Neurochem. 93, 94–104. Ansari, M.A., Ahmad, A.S., Ahmad, M., Salim, S., Yousuf, S., Ishrat, T., Islam, F., 2004. Selenium protects cerebral ischemia in rat brain mitochondria. Biol. Trace Elem. Res. 101, 73–86. Cavalli, R., Caputo, O., Gasco, M.R., 1993. Solid lipospheres of doxorubicin and idarubicin. Int. J. Pharm. 89, R9–R12.

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Please cite this article as: Sachdeva, A.k., et al., Neuroprotective potential of sesamol and its loaded solid lipid nanoparticles in ICV-STZinduced cognitive deficits: Behavioral and biochemical evidence. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.014i