© Borgis - Postępy Fitoterapii 1/2014, s. 3-9
Soma Manasa, *Akondi Butchi Raju
Antiamnesic activity of Solanum melongena L. extract
Department of Pharmacology, Ibn Sina National College for Medical Studies, Jeddah, Kingdom of Saudi Arabia
Solanum melongena L. (egg plant) is proved to contain antioxidant and neuroprotective agents. So we evaluated the antiamnesic activity of the present extract.
Antiamnesic activity was evaluated using scopolamine, an inducing agent and memory disruptor and employed various behavioral and biochemical parameters like radial arm maze test, active avoidance test. Acetylcholinestarase activity in the brain was measured and oxidative stress was also determined.
Dose dependent reduction in the working and reference memory errors were identified in radial arm maze test. Increased active avoidances were reported after treatment with high dose of extract (400 mg/kg) in active avoidance testing. Acetylcholinestarse levels and oxidative stress parameters were maintained normal in extract treated groups and the values are comparable to standard drug piracetam treatment.
Based on the results of behavioral and biochemical studies, hypothesize that egg plant extract may act directly as a free radical scavenger or regulator to inhibit acetylcholinesterase due to the presence of phytoconstituents mainly flavonoids, polyphenols which might be responsible for exhibiting antiamnesic activity.
Memory is the complex process of the brain which involves acquisition of information from the surroundings and consolidation of the acquired information and then retrieving it for future use. Central cholinergic system plays a major role in learning and memory process through various neuronal pathways and neurotransmitters. Deficits occurring in these pathways may result in occurance of various cognitive disorders like amnesia and dementia. Alzheimer’s disease (AD) is one of the most common causes of impaired cognitive functions. Besides reduced cholinergic activity, oxidative stress is also one of the major causes for memory loss in AD. Hence, agents which act by reducing oxidative stress and increased cholinergic activity are found to be useful in treating memory impairments (1).
Solanum melongena L. (egg plant) is a plant native of India and is ranked as one of the top ten vegetables in terms of oxygen free radical scavenging capacity (2). Main constituents of egg plant are phenolic compounds, chlorogenic acid and caffeic acids and they were all established as neuroprotective and antioxidant agents (3). In keeping this view in mind the present investigation was carried out on egg plant (Solanum melongena L.) to evaluate its antiamnesic activity.
Scopolamine, a muscarinic receptor antagonist, is reported to impair long term potentiation (LTP), and hence it serves as experimental model of AD and thereby used as amnesic agent for evaluation of antiamnesic effect of new drugs (4).
Materials and methods
Collection of Plant Material
The proposed plant material of fresh Solanum melongena fruits were collected from Mogilicherla, Warangal district of Andhra Pradesh – India in the month of June. The plant was identified and authenticated by Dr. V. S. Raju, Senior Professor in Department of Botany, Kakatiya University, Warangal, India. The voucher specimen of plant was deposited for further reference.
Preparation of extract
The Solanum melongena fruits were first washed well and the seeds were removed from the fruits. The flesh of the fruit was chopped into small pieces (2-4 cm) and shade dried at room temperature. The dried samples were grounded to powder using a grinder. The dried ground powder was passed through a standard 20 mesh size (particle size < 0.850 mm). Shade dried powder was weighed (500 mg) and placed into 15 ml plastic tubes and 10 ml of 80% methanol was added to it. The mixture was vigorously shaken using a vortex mixer for 2 min, then left in a rotary shaker overnight at ambient temperature to ensure effective extraction. The samples were then centrifuged at 5000 rpm for 15 min and the supernatant was filtered using Whatman filter paper. The residues were then re-extracted two more times with additional 10 ml 80% methanol. All three extracts were combined and concentrated using a rotavapor at reduced temperature and pressure in order to remove the solvent completely. It was dried and kept in a desiccator till experimentation (5).
All experiments were conducted using Albino Wistar rats (150-200 g) of both the sexes at about 6-8 weeks of age. All animals were procured from Sanzyme Ltd., Hyderabad. The animals were maintained with free access to food and water and kept at 25 ±2°C under a controlled 12 h light/dark cycle. The mice were allowed to acclimatize to the laboratory environment for a week before the start of the experiment. The care and maintenance of the animals were carried out as per the approved guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi. The research protocols were approved by the Institutional Animal Ethical Committee (IAEC). The approval number is 13/SPIPS/IAEC/12.
Drugs and chemicals
Scopolamine hydrobromide purchased from Boehringer Ingelheim India, acetylcholine chloride, 5,5-dithio-bis2–nitrobenzoic acid, (Ellman’s reagent), acetyl thiocholine iodide, trichloroacetic acid, thiobarbituric acid (TBA) were purchased from Sigma-Aldrich India, piracetam was purchased from Glaxo Smith Kline India.
Acute toxicity study
The acute toxicity was performed according to the OECD 423 guidelines. The extract at the dose of 5, 50, 300 and 2000 mg/kg body weight, was administered to the rats and they were subsequently observed closely for the first 4 h for any untoward symptoms such as tremors, convulsions, exophthalmus, salivation, diarrhea and lethargy followed by observation for a further 14 days. At the end of the experimental period, the animals were observed for any changes in behavioral pattern and mortality. No toxicity was found in all the 4 doses, so the extract was declared safe up to 2000 mg/kg.
The different successive extracts so obtained were subjected to preliminary phytochemical screening by applying different qualitative testes for phytoconstituents. The extract of Solanum melongena contains alkaloids, carbohydrates, phenolics, flavonoids, tannins, steroids and saponin glycosides. The presence of these phytoconstituents was confirmed by TLC.
Grouping and treatment protocol
Five groups of animals were made, each group consisting of six rats. The following were the groups.
Group 1: Vehicle control; rats received only vehicle.
Group 2: Positive control (PC); rats received only vehicle against scopolamine (1 mg/kg, i.p.) – induced amnesia.
Group 3: Standard drug (STD) piracetam (200 mg/kg, i.p.) treated rats against scopolamine induced amnesia.
Group 4: Extract of Solanum melongena (Test 1) (200 mg/kg, p.o) treated rats against scopolamine induced amnesia.
Group 5: Extract of Solanum melongena (Test 2) (400 mg/kg, p.o) treated rats against scopolamine induced amnesia.
Active avoidance test
Active avoidance test helps to evaluate the associative learning of the animal. The criterion for improved cognitive activity was taken as significant increase in the avoidance response (6).
Evaluation of antiamnesic activity by radial arm maze model
A radial arm maze is used to evaluate working memory in the animals. Each arm (50 x 12 cm) of the eight-arm radial maze extends from an octagonal shaped central hub of 30 cm diameter. The platform is elevated 40 cm above the floor, small black metal cups (3 cm in diameter and 1 cm deep) are mounted at the end of each arm that serve as receptacles for reinforces food (7).
Estimation of acetylcholinesterase activity
The acetylcholinesterase activity was estimated using Ellman’s method (8).
Biochemical estimation of markers of oxidative stress
Biochemical tests were conducted 24 h after 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 phosphate buffer (pH 8). The homogenates (10% w/v) were then centrifuged at 10,000 rpm for 15 min and the supernatant so formed was used for the biochemical estimations.
Estimation of superoxide dismutase
Superoxide dismutase activity in the brain was determined using photo oxidation of o-dianisidine sensitized by riboflavin method (9). The change in absorbance was recorded for 4 min at 460 nm using spectrophotometer.
Estimation of lipid peroxidation (LPO)
The extent of lipid peroxidation in the brain was determined quantitatively by performing the method as described by Ohkawaka (10). The amount of malondialdehyde (MDA) was measured by reaction with thiobarbituric acid at 532 nm using spectrophotometer.
Estimation of Catalase activity
Catalase activity was assessed by the method of Beers and Sizer (11) based on the ability of catalase to oxidize hydrogen peroxide. The change in absorbance was recorded for 3 min at 1 min interval at 240 nm using spectrophotometer.
All experimental groups were composed by 6 animals. The results were presented as the mean ± SEM. Statistical analysis was done by ANOVA followed by Bonferroni’s test. P< 0.05 was considered as statistically significant.
Acute toxicity profile
The rats treated with the extract of Solanum melongena, 5-2.000 mg/kg, p.o., exhibited normal behavior. They were alert, with normal grooming, touch response and pain response. There was no sign of passivity, stereotypy and vocalization. Their motor activity and secretory signs were also normal. The animals showed no signs of depression. Alertness, limb tone and grip strength as well as the gait of the animals were normal. The extract of Solanum melongena was found to be safe up to a dose 2.000 mg/kg in rats.
Effect of Solanum melongena extract on behavioral models
Active avoidance test
In active avoidance test number of avoidances were significantly (p < 0.05) less in scopolamine treated group when compared with the control group (tab. 1 and fig. 1). However treatment with standard drug (piracetam) and extract of Solanum melongena (200 and 400 mg/kg, p.o.) seemed to show the protective effect significantly (p < 0.05) against scopolamine-induced memory impairment by inhibiting the incidence of less number of avoidances. Solanum melongena (400 mg/kg) dose shown more prominent results in increasing the avoidance responses compared to (200 mg/kg) dose.
Table 1. Effect of Solanum melongena extract on active avoidance paradigm against scopolamine induced amnesia.
|Treatment groups (n = 6)||Number of avoidance responses|
|1. Vehicle control||12.50 ± 0.22|
|2. Positive control|| 5.83 ± 0.30*|
|3. Standard|| 11.17 ± 0.16#|
|4. Test 1|| 8.50 ± 0.22#|
|5. Test 2|| 9.67 ± 0.21#|
|*P < 0.05 compared with normal control group. #p < 0.05 compared with scopolamine treated group. The results were presented as the mean ± SEM. Statistical analysis was done by ANOVA followed by Bonferroni’s test. P < 0.05 was considered as statistically significant. Values are presented as mean ± SEM, n = 6.|
Fig. 1. Effect of Solanum melongena extract on active avoidance paradigm against scopolamine induced amnesia.
Working memory errors
In radial arm maze, working memory errors were more in scopolamine treated group when compared to control group and indicates memory impairment. While pretreatment with Solanum melongena (200 and 400 mg/kg, p.o.) and piracetam (200 mg/kg, i.p.) there was significant (p < 0.05) reduction in working memory errors when compared to positive control group as shown in table 2 and figure 2. Higher dose of Solanum melongena (400 mg/kg) dose has shown more prominent results in reducing the occurance of working memory errors.
Reference memory errors
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