PHCOG MAG.
ORIGINAL ARTICLE
Sesamol, a lipid lowering agent, ameliorates aluminium chloride induced behavioral and biochemical alterations in rats Jessy John, Madhavan Nampoothiri, Nitesh Kumar, Jayesh Mudgal, Gopalan Kutty Nampurath, Mallikarjuna Rao Chamallamudi Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka, India Submitted: 21-06-2014
Revised: 10-08-2014
Published: 12-03-2015
ABSTRACT
Access this article online
Background: Sesame oil from the seeds of Sesamum indicum Linn. (Pedaliaceae) has been used traditionally in Indian medical practice of Ayurveda in the treatment of central nervous system disorders and insomnia. A few published reports favor the anti‑dementia effect of sesamol (SML), an active constituent of sesame oil. Objective: Thus, the present study was aimed to explore the anti‑dementia effect and possible mechanism (s) of SML in aluminium chloride (AlCl3)‑induced cognitive dysfunction model in rodents with special emphasis on memory centers viz., hippocampus and frontal cortex. Methods: Male Wistar rats were exposed to AlCl3 (175 mg/kg p.o.) for 60 days. SML (10 and 20 mg/kg) and rivastigmine (1 mg/kg) were administered orally 45 min before administration of AlCl3 for 60 days. Spatial memory was assessed using Morris water maze test. After 60 days of treatment animals were sacrificed, hippocampus and frontal cortex were collected and analyzed for acetylcholinesterase (AChE) activity, tumor necrosis factor (TNF‑α) level, antioxidant enzymes (Glutathione, catalase), lipid peroxidation, and nitrite level. The circulating triglycerides, total cholesterol, low‑density lipoprotein (LDL) and high‑density lipoprotein (HDL) levels were also analyzed. Results: SML significantly prevented behavioral impairments in aluminium‑exposed rats. Treatment with SML reversed the increased cholesterol, triglycerides and LDL while raised the HDL levels. SML significantly corrected the effect of AlCl3 on AChE activity. Further, SML reversed the elevated nitric oxide, TNF‑α and reduced antioxidant enzymes in hippocampus and frontal cortex. Conclusion: The present study suggests the neuro‑protection by SML against cognitive dysfunction induced by environmental toxin (AlCl3) in hippocampus and frontal cortex.
Website: www.phcog.com DOI: 10.4103/0973-1296.153086 Quick Response Code:
Key words: Aluminium, dementia, hypolipidemia, memory, sesamol, tumor necrosis factor‑α
INTRODUCTION Aluminium, a highly neurotoxic metal, is considered to be involved in the pathogenesis of neurodegenerative disorders like Alzheimer’s disease (AD)[1‑3] and Parkinson’s disease.[4] Experimental animals, exposed to aluminium have developed AD‑like conditions, characterized by elevated levels of amyloid beta (Aβ) protein and amyloid precursor protein (APP),[5,6] mitochondrial dysfunction, depletion of ATP,[7,8] induction of lipid peroxidation and lipid dystrophy, [9,10] accelerated production of phosphorylated tau, [11] impairment of cholinergic Address for correspondence: Dr. C. Mallikarjuna Rao, Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal ‑ 576 104, Karnataka, India. E‑mail: [email protected] Pharmacognosy Magazine | April-June 2015 | Vol 11 | Issue 42
projections[12] and promotion of apoptotic neuronal death.[13,14] Thus, aluminium chloride (AlCl3)‑induced cognitive dysfunction model has been widely used for testing drugs against AD.[15‑17] Cur rently approved treatments for AD targ et neurotransmitter systems and only provide modest improvement in cognitive impairment. Thus, it is necessary to develop effective medications that go beyond acetylcholinesterase (AChE) inhibitors and N‑methyl‑D‑aspartate antagonist. Several studies have demonstrated that hypercholesterolemia could cause dementia and Aβ42 deposition in hippocampal region.[18] Hypercholesterolemia is an outcome of sedentary life‑style resulting in obesity and lipid dystrophy. Traditionally, in Indian medical practice of Ayurveda, sesame oil from the seeds of Sesamum indicum Linn. (Pedaliaceae) has been used to correct central nervous system disorders and insomnia.[19] 327
John, et al.: Neuroprotective role of sesamol
Sesamol (SML), an agent obtained from sesame oil, edible oil, is found to reduce cholesterol and triglyceride levels in acute and chronic models of hyperlipidemia.[20] It is also reported to have antioxidant, neuro‑protective,[21] anti‑inflammatory[22] and hepatoprotective[23] activities. Although, all these actions of SML are beneficial to overcome the condition of dementia, SML has not been investigated for its behavioral effects in chronic models of dementia. In view of this, the present study was designed to investigate the effect of SML, a lipid lowering agent in AlCl3‑mediated behavioral and biochemical changes in rats.
MATERIALS AND METHODS Animals
Male Wistar rats, weighing 200–250 g (90 days old) procured from Central Animal Research Facility of Manipal University, Manipal were used. Animals were acclimatized to laboratory conditions for 7 days before the experiment and they were maintained under controlled conditions of temperature (23°C ± 2°C), humidity (50% ±5%). The animals were kept under standard conditions of 12 h light/dark cycle in sanitized polypropylene cages containing sterile paddy husk as bedding with free access to food and water ad libitum. The experimental protocol was approved by the Institutional Animal Ethics Committee, Kasturba Medical College, Manipal [IAEC/KMC/73/2012] and was carried out in accordance with the guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India. Drugs and treatment schedule
Aluminium chloride (Spectrochem Pvt Limited, India), SML (Sigma‑Aldrich Co, St. Louis, MO, USA) and rivastigmine (RIV) (Dr. Reddy’s Laboratories, Hyderabad, India) solutions were made freshly on each day for administration. AlCl3 was dissolved in distilled water and administered orally once daily at a dose of 175 mg/kg from day 6 onwards (24 h after the completion of retention trial on day 5) for 60 days. This dosing regimen for inducing dementia using AlCl3 was determined according to the previous reports and the high rate of induction and low mortality, which was evident in the pilot study conducted. SML and RIV, at various doses, were administered 45 min before administration of AlCl3 orally after suspending them in 0.5% sodium carboxy methyl cellulose (CMC) in distilled water for 60 days from day 6. On the basis of escape latency time (ELT) on day 5, animals were divided into six groups (n = 8). The groups were as follows: Group 1: N o r m a l c o n t r o l ‑ Re c e ive d d i s t i l l e d water (5 ml/kg p.o.). Group 2: Vehicle control ‑ Receives 0.5% CMC (5 ml/kg p.o.). Group 3: AlCl3 (175 mg/kg p.o.). 328
Group 4: RIV (1 mg/kg p.o.) + AlCl3 (175 mg/kg p.o.). Group 5: SML (10 mg/kg p.o.) + AlCl3 (175 mg/kg p.o.). Group 6: SML (20 mg/kg p.o.) + AlCl3 (175 mg/kg p.o.). The doses of the standard drug RIV (1 mg/kg) and the test drug SML (10 mg/kg and 20 mg/kg) were chosen based on the previous literature reports.[24‑27] Body weight of the animals was taken on a daily basis before the treatments. Spatial memory assessment using Morris water maze
To investigate the spatial learning and memory abilities of the experimental rats, Morris water maze task was performed as described by Morris[28] with minor modifications.[29] It consisted of a circular tank of 150 cm diameter and 40 cm height. The pool was divided into North‑East, South‑East, South‑West and North‑West (NW) quadrants. In the NW quadrant a hidden escape platform (10 cm diameter), was placed 2 cm below the water surface. All rats were trained to find the escape platform. Animals were given four trials per day for 4 consecutive days. Animals were kept on the platform for 30 s and then removed. The rats that could not reach the platform in 20 s on the 4th trial‑day were excluded from the study. On the probe day (day 5), the hidden platform was removed, and probe trial was performed with a cut off time of 60 s. All the animals were exposed to one retention trial on day 25, 45 and 65 to evaluate the memory consolidation. Data were acquired through a video camera connected to a computerized tracking system (Any Maze, Ugo Basile, Italy) fixed above the centre of the pool. Dissection and tissue preparation
On day 65, immediately after the retention trial, the animals were sacrificed by decapitation. Brains were rapidly removed, hippocampus and frontal cortex were dissected according to the method described by Glowinski and Iverson.[30] A 10% w/v homogenate of samples was prepared by homogenizing with ice‑cooled 0.1 M phosphate buffer potential of Hydrogen (pH) 7.4 using an ultra Turrax T25 homogenizer at a speed of 9500 rpm thrice at an interval of few seconds. The homogenates were then centrifuged at 15,000 rpm at 4°C for 15 min. Supernatant was collected and used for biochemical estimations. Estimation of acetylcholinesterase activity
In the supernatant, AChE activity was measured by Ellman method using acetylthiocholine iodide as a substrate.[31] To a reaction mixture containing phosphate buffer (2.8 ml, pH 8), acetylthiocholine iodide (0.05 ml) and 0.05 ml of 5,5’‑dithio‑bis‑2‑nitrobenzoic acid (DTNB) (Ellman reagent), 0.1 ml of the supernatant was added. The change in absorbance was measured for 4 min at 60 s interval at Pharmacognosy Magazine | April-June 2015 | Vol 11 | Issue 42
John, et al.: Neuroprotective role of sesamol
412 nm using ultraviolet–visible spectrophotometer and the change in absorbance per minute was calculated. The results were expressed as micromoles of acetylthiocholine iodide hydrolyzed per min per mg protein.
Pad Prism version 5.0 software. P
ORIGINAL ARTICLE
Sesamol, a lipid lowering agent, ameliorates aluminium chloride induced behavioral and biochemical alterations in rats Jessy John, Madhavan Nampoothiri, Nitesh Kumar, Jayesh Mudgal, Gopalan Kutty Nampurath, Mallikarjuna Rao Chamallamudi Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka, India Submitted: 21-06-2014
Revised: 10-08-2014
Published: 12-03-2015
ABSTRACT
Access this article online
Background: Sesame oil from the seeds of Sesamum indicum Linn. (Pedaliaceae) has been used traditionally in Indian medical practice of Ayurveda in the treatment of central nervous system disorders and insomnia. A few published reports favor the anti‑dementia effect of sesamol (SML), an active constituent of sesame oil. Objective: Thus, the present study was aimed to explore the anti‑dementia effect and possible mechanism (s) of SML in aluminium chloride (AlCl3)‑induced cognitive dysfunction model in rodents with special emphasis on memory centers viz., hippocampus and frontal cortex. Methods: Male Wistar rats were exposed to AlCl3 (175 mg/kg p.o.) for 60 days. SML (10 and 20 mg/kg) and rivastigmine (1 mg/kg) were administered orally 45 min before administration of AlCl3 for 60 days. Spatial memory was assessed using Morris water maze test. After 60 days of treatment animals were sacrificed, hippocampus and frontal cortex were collected and analyzed for acetylcholinesterase (AChE) activity, tumor necrosis factor (TNF‑α) level, antioxidant enzymes (Glutathione, catalase), lipid peroxidation, and nitrite level. The circulating triglycerides, total cholesterol, low‑density lipoprotein (LDL) and high‑density lipoprotein (HDL) levels were also analyzed. Results: SML significantly prevented behavioral impairments in aluminium‑exposed rats. Treatment with SML reversed the increased cholesterol, triglycerides and LDL while raised the HDL levels. SML significantly corrected the effect of AlCl3 on AChE activity. Further, SML reversed the elevated nitric oxide, TNF‑α and reduced antioxidant enzymes in hippocampus and frontal cortex. Conclusion: The present study suggests the neuro‑protection by SML against cognitive dysfunction induced by environmental toxin (AlCl3) in hippocampus and frontal cortex.
Website: www.phcog.com DOI: 10.4103/0973-1296.153086 Quick Response Code:
Key words: Aluminium, dementia, hypolipidemia, memory, sesamol, tumor necrosis factor‑α
INTRODUCTION Aluminium, a highly neurotoxic metal, is considered to be involved in the pathogenesis of neurodegenerative disorders like Alzheimer’s disease (AD)[1‑3] and Parkinson’s disease.[4] Experimental animals, exposed to aluminium have developed AD‑like conditions, characterized by elevated levels of amyloid beta (Aβ) protein and amyloid precursor protein (APP),[5,6] mitochondrial dysfunction, depletion of ATP,[7,8] induction of lipid peroxidation and lipid dystrophy, [9,10] accelerated production of phosphorylated tau, [11] impairment of cholinergic Address for correspondence: Dr. C. Mallikarjuna Rao, Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal ‑ 576 104, Karnataka, India. E‑mail: [email protected] Pharmacognosy Magazine | April-June 2015 | Vol 11 | Issue 42
projections[12] and promotion of apoptotic neuronal death.[13,14] Thus, aluminium chloride (AlCl3)‑induced cognitive dysfunction model has been widely used for testing drugs against AD.[15‑17] Cur rently approved treatments for AD targ et neurotransmitter systems and only provide modest improvement in cognitive impairment. Thus, it is necessary to develop effective medications that go beyond acetylcholinesterase (AChE) inhibitors and N‑methyl‑D‑aspartate antagonist. Several studies have demonstrated that hypercholesterolemia could cause dementia and Aβ42 deposition in hippocampal region.[18] Hypercholesterolemia is an outcome of sedentary life‑style resulting in obesity and lipid dystrophy. Traditionally, in Indian medical practice of Ayurveda, sesame oil from the seeds of Sesamum indicum Linn. (Pedaliaceae) has been used to correct central nervous system disorders and insomnia.[19] 327
John, et al.: Neuroprotective role of sesamol
Sesamol (SML), an agent obtained from sesame oil, edible oil, is found to reduce cholesterol and triglyceride levels in acute and chronic models of hyperlipidemia.[20] It is also reported to have antioxidant, neuro‑protective,[21] anti‑inflammatory[22] and hepatoprotective[23] activities. Although, all these actions of SML are beneficial to overcome the condition of dementia, SML has not been investigated for its behavioral effects in chronic models of dementia. In view of this, the present study was designed to investigate the effect of SML, a lipid lowering agent in AlCl3‑mediated behavioral and biochemical changes in rats.
MATERIALS AND METHODS Animals
Male Wistar rats, weighing 200–250 g (90 days old) procured from Central Animal Research Facility of Manipal University, Manipal were used. Animals were acclimatized to laboratory conditions for 7 days before the experiment and they were maintained under controlled conditions of temperature (23°C ± 2°C), humidity (50% ±5%). The animals were kept under standard conditions of 12 h light/dark cycle in sanitized polypropylene cages containing sterile paddy husk as bedding with free access to food and water ad libitum. The experimental protocol was approved by the Institutional Animal Ethics Committee, Kasturba Medical College, Manipal [IAEC/KMC/73/2012] and was carried out in accordance with the guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India. Drugs and treatment schedule
Aluminium chloride (Spectrochem Pvt Limited, India), SML (Sigma‑Aldrich Co, St. Louis, MO, USA) and rivastigmine (RIV) (Dr. Reddy’s Laboratories, Hyderabad, India) solutions were made freshly on each day for administration. AlCl3 was dissolved in distilled water and administered orally once daily at a dose of 175 mg/kg from day 6 onwards (24 h after the completion of retention trial on day 5) for 60 days. This dosing regimen for inducing dementia using AlCl3 was determined according to the previous reports and the high rate of induction and low mortality, which was evident in the pilot study conducted. SML and RIV, at various doses, were administered 45 min before administration of AlCl3 orally after suspending them in 0.5% sodium carboxy methyl cellulose (CMC) in distilled water for 60 days from day 6. On the basis of escape latency time (ELT) on day 5, animals were divided into six groups (n = 8). The groups were as follows: Group 1: N o r m a l c o n t r o l ‑ Re c e ive d d i s t i l l e d water (5 ml/kg p.o.). Group 2: Vehicle control ‑ Receives 0.5% CMC (5 ml/kg p.o.). Group 3: AlCl3 (175 mg/kg p.o.). 328
Group 4: RIV (1 mg/kg p.o.) + AlCl3 (175 mg/kg p.o.). Group 5: SML (10 mg/kg p.o.) + AlCl3 (175 mg/kg p.o.). Group 6: SML (20 mg/kg p.o.) + AlCl3 (175 mg/kg p.o.). The doses of the standard drug RIV (1 mg/kg) and the test drug SML (10 mg/kg and 20 mg/kg) were chosen based on the previous literature reports.[24‑27] Body weight of the animals was taken on a daily basis before the treatments. Spatial memory assessment using Morris water maze
To investigate the spatial learning and memory abilities of the experimental rats, Morris water maze task was performed as described by Morris[28] with minor modifications.[29] It consisted of a circular tank of 150 cm diameter and 40 cm height. The pool was divided into North‑East, South‑East, South‑West and North‑West (NW) quadrants. In the NW quadrant a hidden escape platform (10 cm diameter), was placed 2 cm below the water surface. All rats were trained to find the escape platform. Animals were given four trials per day for 4 consecutive days. Animals were kept on the platform for 30 s and then removed. The rats that could not reach the platform in 20 s on the 4th trial‑day were excluded from the study. On the probe day (day 5), the hidden platform was removed, and probe trial was performed with a cut off time of 60 s. All the animals were exposed to one retention trial on day 25, 45 and 65 to evaluate the memory consolidation. Data were acquired through a video camera connected to a computerized tracking system (Any Maze, Ugo Basile, Italy) fixed above the centre of the pool. Dissection and tissue preparation
On day 65, immediately after the retention trial, the animals were sacrificed by decapitation. Brains were rapidly removed, hippocampus and frontal cortex were dissected according to the method described by Glowinski and Iverson.[30] A 10% w/v homogenate of samples was prepared by homogenizing with ice‑cooled 0.1 M phosphate buffer potential of Hydrogen (pH) 7.4 using an ultra Turrax T25 homogenizer at a speed of 9500 rpm thrice at an interval of few seconds. The homogenates were then centrifuged at 15,000 rpm at 4°C for 15 min. Supernatant was collected and used for biochemical estimations. Estimation of acetylcholinesterase activity
In the supernatant, AChE activity was measured by Ellman method using acetylthiocholine iodide as a substrate.[31] To a reaction mixture containing phosphate buffer (2.8 ml, pH 8), acetylthiocholine iodide (0.05 ml) and 0.05 ml of 5,5’‑dithio‑bis‑2‑nitrobenzoic acid (DTNB) (Ellman reagent), 0.1 ml of the supernatant was added. The change in absorbance was measured for 4 min at 60 s interval at Pharmacognosy Magazine | April-June 2015 | Vol 11 | Issue 42
John, et al.: Neuroprotective role of sesamol
412 nm using ultraviolet–visible spectrophotometer and the change in absorbance per minute was calculated. The results were expressed as micromoles of acetylthiocholine iodide hydrolyzed per min per mg protein.
Pad Prism version 5.0 software. P
