LEARNING AND MEMORY ENHANCING ACTIVITY OF ETHANOLIC EXTRACT OF DRIED SEED KERNELS OF Caesalpinia crista Lin. IN ALBINO MICE

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1 Available online at ISSN Research Article PHARMACIE GLOBALE INTERNATIONAL JOURNAL OF COMPREHENSIVE PHARMACY LEARNING AND MEMORY ENHANCING ACTIVITY OF ETHANOLIC EXTRACT OF DRIED SEED KERNELS OF Caesalpinia crista Lin. IN ALBINO MICE Sunil N Kshirsagar* 1, Dinesh M Sakarkar 2 and Supriya S Deshpande 3 1 Department of Pharmacology, Sudhakarrao Naik Institute of Pharmacy, Pusad, Maharashtra, India. 2 Principal, Sudhakarrao Naik Institute of Pharmacy, Pusad, Maharashtra, India. 3 Department of Pharmacology, Dr P D Medical College Amaravati, Maharashtra, India. Received: 21 June 2012; Revised: 12 August 2012; Accepted: 27 August 2012; Available online: 5 September 2012 ABSTRACT Dementia is one of the age related mental problem and characteristic symptoms of various neurodegenerative disorder including Alzheimer s disease. Stressful conditions are often associated with loss of memory and other cognitive functions. Various drugs like diazepam, alcohol, barbiturates disrupt learning and memory. Traditionally herbal drugs have been used to enhance cognitive function. Present work was undertaken to assess the potential of dried seed kernels of Ceasalpinia crista extract as learning and memory enhancer. Ethanolic extract of dried seed kernels of Ceasalpinia crista linn.ameliortated the amnesic effect of scopolamine in mice. Redial arm maze paradigm served as the exteroceptive behavioral models. Ethanolic extract of dried seed kernels of Caesalpinia crista Lin. is compared with standard drug piracetam in scopolamine induced amnesia in mice. Estimation of Acetyl Choline Esterase (AChE) activity provides a relatively easy and valuable assessment of cholinergic function. A decrease in AChE activity was noted in the treated animals leading to increased cholinergic activity in the brain. There was significant decrease in the AChE activity assayed from hypothalamus, frontal cortex and hippocampus of the mice brain, treated with Ethanolic extract of dried seed kernels of Caesalpinia crista Lin. No side effects were observed with administration of the Ethanolic extract of dried seed kernels of Caesalpinia crista Lin. Keywords: Acetyl choline esterase; learning Memory; Ceasalpinia crista; Piracetam; Radial arm maze. INTRODUCTION Plants used in traditional medicine contain wide range of ingredients that can be used to treat chronic as well as infectious diseases. A vast knowledge of how to use the plants against different illness may be expected to have accumulated in areas where the use of plants is still of great importance. 1 The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. The important constitutents of these plants are alkaloids, Flavonoids, tannins and phenolic compounds. 2 Caesalpinia crista (Lin.) synonym: Caesalpinia bonduc (L.) Roxb.), Caesalpinia bonducella belongs to family Caesalpiniaceae and is commonly known as kat-takaranja in Hindi and sagargota in Marathi. It is prickly shrub found throughout the hotter regions of India and srilanka. 3 The seeds of plant are almost globular in shape, grey, hard with a smooth shiny surface. 4 The seeds of the plant contain bonducin, proteins, saponin, starch, sucrose, an enzyme, two phytosterols namely sitosterol and hepatsane, fattyacids such as palmitic acid, stearic acid, lognoceric, oleic, linolenic acid. The seed kernals of the plant contain furanoditerpenes- ά-caesalpin, ß-caesalpin, γ-caesalpin, δ-caesalpin, ε-caesalpin,and F-caesalpin. 5 The *Corresponding Author: Mr Sunil N Kshirsagar Atiyhya Shankar Nagar, Pusad , Dist- Yavatamal, Maharashtra, India. Contact no: ; sunilkshi123@rediffmail.com leaves of Caesalpinia crista (Lin) are tradionally used for tumors, inflammation and liver disorders. In India, various parts of this plant has been used in various therapeutic uses like adaptogenic 6, antimicrobial 7, antiproliferatve 8, antidiabetic 9, anti-filarial 10, contractility on uterus 11, hepatprotective 12, antitumor and antioxidant activities 13. Because of various pharmacological activities, present work was undertaken to study the learning and memory enhancing activity of ethanolic extract of seed kernels of Caesalpinia crista Lin. in albino mice. MATERIALS AND METHODS Plant Material The Caesalpinia crista (Lin.) seeds were collected from the local market Yogesh Pharmacy Nanded Maharastra. It was authenticated by Dr Mr Krishna G Kadaskar, Department of Botany P N College, Pusad (Maharastra). Preparation of Extract The seeds kernels of Caesalpinia Crista (Lin.) were shade dried at room temperature. Then the shade dried seeds kernels were powdered to get a coarse powder. 100g of coarse powder was defatted with petroleum ether and extracted exhaustively with 95% ethanol at Temperature 60 C, in a soxhlet extractor. The extract was concentrated in a rotary flash evaporator residue was dried in desiccator over sodium sulfite. This procedure was repeated for 5-6 times to receive sufficient quantity of 1 Pharmacie Globale (IJCP), Vol. 03, Issue 09

2 ethanolic extract. 14 Phytochemical Investigation Ethanolic extract of Caesalpinia crista (Lin.) seeds kernels were subjected to further preliminary qualitative phytochemical investigation. 15 Experimental Animals Healthy adult Albino mice of either sex weighing gm acclimatized for 14 days. The animals were housed under standard conditions and room temperature (25±2 C). During the acclimatization period of 14 days, animals were observed for general condition every day and weighed on the next day of arrival and on the last day of acclimatization. The animals were fed with balanced pellet and water adlibitum. The experimental protocol of toxicological studies was reviewed and approved by the Institutional Animal Ethical Committee of Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA). Drugs and chemicals Test drug: Caesalpinia crista Lin dried seed kernel ethanolic extract; Dose: 200mg/kg, 300 mg/kg and 400mg/kg body weight given orally for 14 days. Standard drug: Piracetam in water for injection was prepared as per requirement; Dose: 150 mg/kg i.p. Grouping and treatment protocol Five groups of animals were made, each group consisting of six mice. The following were the groups. Group 1: Normal control mice received only vehicle. Group 2: Ethanolic extract of Caesalpinia crista Lin. dried seeds, 100mg/kg p.o. treated mice. Group 3: Ethanolic extract of Caesalpinia crista Lin. dried seeds, 200mg/kg p.o. treated mice. Group 4: Ethanolic extract of Caesalpinia crista Lin. dried seeds, 400mg/kg p.o. treated mice. Group 5: Standard drug Piracetam, 150mg/kg i.p. treated mice. The animals were divided into five groups containing six animals each. The first groups was Normal control group, second, third and fourth were pre-treated with 100mg, 200mg and 400 mg/kg of body weight the ethanolic extract of dried seed kernel of Caesalpinia crista Lin. and fifth groups was pre-treated with standard drug Piracetam (150mg/kg) i.p for 14 days respectively. The animals were trained for two consecutive days and their behavioural changes were studied for 16 days. Assessment of behaviour in the radial arm maze The eight arm radial maze consisted of equally spaced arms (42 x 1.4 x 1.4cm) radiating from an octagonal central platform and the maze was kept 80cm elevated from the ground. Prior to the training, the animals were kept on a restricted diet and body weight was maintained at 85% of their free feeding weight, with water available ad-libitum. Before the commencement of the behavioural assessment, all groups of mice were semi-starved for 48 hrs in order to motivate them towards food reward. On subsequent days, food was provided to maintain 85% of its initial body weight, throughout experimental period. Training To acclimatize the mice to the radial arm maze prior to acquisition all the arms were baited and mice were allowed to explore the maze for 10 minutes and were given two such sessions on two consecutive days. trained to choose only the baited arms. This task permits discerning of reference memory and working memory components of spatial memory. An entry into an unbaited arm is regarded as a reference memory error and any reentry either to a baited or unbaited arm is considered as a working memory error. Acquisition Mice were given two trials per day. At the beginning of each trial, the maze was thoroughly cleaned with 70% ethanol and four of the arms (2, 3, 6 and 8) were baited with food reinforcement. The mice was placed in the centre of the octagon and allowed a free choice. An arm choice was recorded when a mice ate a bait or reached the end of an arm. The maze arms were not rebaited, so only the first entry into the baited arm was recorded as a correct choice. The trial continued until the mice entered all the four baited arms or 5 minutes had elapsed. At the end of the trial, the mice were returned to the home cages and were subjected to second trial after an inter-trial interval of l hour. Training was continued till the mice attained the criteria of 80% correct choice. Evaluation criteria Data from 4 trials were averaged and represented as blocks. The data was analyzed for percent correct choice, reference and working memory errors. Percent correct choice was calculated by dividing the number of correct entries by the total number of entries and multiplying by 100. An entry into an unbaited arm was considered a reference memory error and any re-entry was considered as a working memory error. Assay of acetylcholinesterase (AChE) activity in the brain After behavioural assessment on these tasks, the alteration in the AChE activity was evaluated in the frontal cortex, hippocampus, septum, hypothalamus and brain stem. Mice were decapitated and the brain regions were quickly dissected out on a chilled petridish. AChE activity was determined using Ellman s method 16 with minor modifications as described elsewhere. 17 The esterase activity was measured by providing the substrate, acetylthiocholine (ATC). Thiocholine released by the cleavage of ATC by AChE is allowed to react with the -SH reagent 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB), which is reduced to thionitrobenzoic acid, a yellow coloured anion with an absorption maxima at 412nm.The extinction co-efficient of the thionitro benzoic acid is 1.36 x104/mole/centimeter The concentration of thionitro benzoic acid detected using a UV spectrophotometer is then taken as a direct estimate of the AChE activity. RESULT AND DISCUSSION Behavioural Study The radial arm maze has been widely used in behavioural neuroscience. Thus radial arm maze tests are useful in evaluating the effect of drugs, stress and various other environmental factors on learning and memory. 18 Reference memory and working memory are the two variables that report the physiological status of the brain. Amongst the various functions of the brain, one of the most interesting one is the ability to acquire new information and store them for further retrieval. Learning is a process of acquiring new information, while Memory refers to the persistence of a change in behavior overtime, in a state that can be retrieved later. The effect of ethanolic extract of dried seed kernel of Caesalpinia crista Lin. on performance of a partially baited radial arm maze task was Partially baited radial arm maze task In this, four of the eight arms are baited and the mice are 2 Pharmacie Globale (IJCP), Vol. 03, Issue 09

3 studied. Results on the correct choice of a partially baited radial arm maze task are given in the table. The data in the table 1 clearly shows that the % of correct choice of group- 1 animals was ± 2.10 in the first block of 4 trials and as the trials increased the % correct choice goes up to ± 1.95, i.e. almost 2 fold and the increase of % correct choice is in arithmetic progression as the number of trial increases. Results of this experiment reveal that as the trials are increased the % correct choice also increased (almost parallel) and hence the radial arm maze experiments could be reliably used for our actual study that is alleviating role of ethanolic extract of dried seed kernel of Caesalpinia crista Lin. in stress animals. Observations of the effect of ethanolic extract of dried seed kernel of Caesalpinia crista Lin. to normal mice on reference memory errors and working memory errors (correct) in partially baited radial arm maze task are expressed in tables 2 and 3 respectively. Table 1. Effect of ethanolic extract of dried seed kernel of Caesalpinia crista Lin. in normal mice and performance in a partially baited radial arm maze task ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 2.61 Data is represented as Mean ± SEM. Group 1 was normal control, Group 2, 3, 4 was subjected to 14 days of treatment with ethanolic extract of dried seed kernel of Caesalpinia crista Lin. 100, 200 and 400 mg/kg, p.o. respectively. Group 5 was treated with Standard drug Piracetam (150mg/kg) i.p. Table 2. Effect of Ethanolic extract of dried seed kernel of Caesalpinia crista Linn.in normal mice on reference memory errors in partially baited radial arm maze task ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.16 Data is represented as Mean ± SEM. Group 1 was normal control, Group 2, 3, 4 was subjected to 14 days of treatment with ethanolic extract of dried seed kernel of Caesalpinia crista Lin. 100, 200 and 400 mg/kg, p.o., respectively. Group 5 was treated with Standard drug Piracetam (150mg/kg) i.p. Table 3. Effect of ethanolic extract of dried seed kernel of Caesalpinia crista Lin. in normal mice on working memory errors in partially baited radial arm maze task ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.16 Data is represented as Mean ± SEM. Group 1 was normal control,group was subjected to 14 days of treatment with ethanolic extract of dried seed kernel of Caesalpinia crista Linn 100,200 and 400 mg/kg, p.o., respectively. Group 5 was treated with Standard drug Piracetam (150mg/kg) i.p. The data in table 2 shows a significant decrease in reference memory errors as trials progressed. In Group-1 animals the reference memory error is 3.93 ± 0.23 in the first block of 4 trials and as trials increased the reference memory error is brought down to 0.55 ± 0.09 in the eighth block of four trials. Animals treated with ethanolic extract of dried seed kernel of Caesalpinia crista Lin. to Group-2, Group-3, Group-4 also showed a similar trend. But the treated animals did not show a greater decrease than control animals. Group-2, Group-3, Group-4 animals were compared with Group-5 animals which were treated with Standard drug Piracetam (150mg/kg), in which Group-4 animals were show significant enhancement in learning and memory. The table 3 which gives the data on working memory error also showed a decrease in working memory error as trials progressed. This trend was also observed in animals Group-2, Group-3, Group-4. But this decrease in treated animals was not appreciable compared to normal control. In the present study the higher dose 400mg/kg /day for 14 days showed better performance than 100 and 200 mg/kg doses. Incidentally chronic administration of ethanolic extract of dried seed kernel of Caesalpinia crista Lin. was not show side effects in animals even with the 400 mg/kg dose regimen. Chronic treatment with 400 mg/kg ethanolic extract of dried seed kernel of Caesalpinia crista Lin. alone resulted in learning and memory enhancement. Biochemical study Estimation of acetylcholinesterase (AChE) activity provides a relatively easy and valuable assessment of cholinergic function. In the recent past, the role of ACh in learning and memory has been demonstrated indubitably. Thus assessing cholinergic function is considered as an important tool in neuroscience research. Acetylcholine per se has a very short half-life and direct estimation of ACh is a little difficult in brain homogenates. There are several approaches to evaluate cholinergic function indirectly. Estimating the expression of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE) by immunochemical and histochemical techniques provide information on the cholinergic function, but are tedious 3 Pharmacie Globale (IJCP), Vol. 03, Issue 09

4 and time consuming. Estimation of AChE activity provides a relatively easy and valuable assessment of cholinergic function. The table 4 shows the AChE activity in the five brain regions namely frontal cortex, hippocampus, septum, hypothalamus and brain stem. Table 4. AChE data table Regions Group-1 Group-2 Group-3 Group-4 Group-5 Frontal cortex 5.22 ± ± ± ± ± 0.13 Hippocampus 5.32 ± ± ± ± ± 0.18 Septum 9.77 ± ± ± ± ± 0.63 Hypothalamus 6.24 ± ± ± ± ± 0.26 Brainstem 7.11 ± ± ± ± ± 0.23 Data is represented as Mean ± SEM. Group 1 was normal control,group was subjected to 14 days of treatment with ethanolic extract of dried seed kernel of Caesalpinia crista Linn 100,200 and 400 mg/kg, p.o., respectively Group 5 was treated with Standard drug Piracetam (150mg/kg) i.p. The results clearly show that AChE activity showed a gradual decrease in frontal cortex when treated with an increasing dose of ethanolic extract of dried seed kernel of Caesalpinia crista Lin. There is a marginal increase in activity in the hippocampus when the animal is treated with ethanolic extract of dried seed kernel of Caesalpinia crista Lin. 200 mg/kg. There is enhanced AChE activity in the septum when treated with 100 and 400 mg/kg body weight of the mice while a decline in activity at higher dose in the hypothalamus is seen. An increase in AChE activity in the brain stem in 200 mg and 400 mg was observed. At higher dose of ethanolic extract of dried seed kernel of Caesalpinia crista Lin. did appear to inhibit brain cholinesterase activity in the hippocampus and hypothalamus without affecting in frontal cortex. In the least, these data may provide the impetus for further study of the material, and isolation of its active components. The mechanism of action by which enhances learning and memory performance in behavioural tasks is as yet unknown. At higher dose, ethanolic extract of dried seed kernel of Caesalpinia crista Lin. did appear to inhibit brain cholinesterase activity in the hippocampus and hypothalamus without affecting in frontal cortex. Furthermore, this plant may be more effective in individuals who are cognitively impaired as a result of chemical or organic brain damage as compared with normal subjects. In the least, these data may provide the impetus for further study of the material, and isolation of its active components. Studies on brains from patients suffering from Alzheimer s disease have shown reduced AChE activity in the hippocampus and cortex. 19 CONCLUSION This study demonstrates that ethanolic extract of dried seed kernel of Caesalpinia crista Lin. enhances learning and memory activity, impairment induced by scopolamine and these effects are mediated in part by inhibition of AChE activity in the brain. 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