|Year : 2019 | Volume
| Issue : 1 | Page : 41-46
Effect of ethanolic extract of leaves of Solanum trilobatum on scopolamine-induced memory impairment in Sprague Dawley rats
Subramani Parasuraman, Siaw Sze Hoong, Parayil Varghese Christapher, Lim Nian Zou, David Lim De Wei, Sunderesan Loshini, Teoh Huey Ching, Chong Hao Leong
Unit of Pharmacology, Faculty of Pharmacy, AIMST University, Bedong, Kedah, Malaysia
|Date of Web Publication||22-Aug-2019|
Unit of Pharmacology, Faculty of Pharmacy, AIMST University, Bedong 08100, Kedah
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: The objective of the study is to determine the effect of ethanolic extract of leaves of Solanum trilobatum (EEST) on scopolamine-induced memory impairment in Sprague Dawley (SD) rats. Materials and Methods: The leaves of S. trilobatum were extracted with ethanol and used for the study. The SD rats were divided into six groups each consist of six animals, namely normal control; scopolamine control; donepezil treated 3 mg/kg; and EEST treated 100, 200, and 400 mg/kg, respectively. Except normal control, all other animals were administered with intraperitoneal injection of scopolamine (1 mg/kg body weight) for 21 days to induce memory impairment. Donepezil and EEST were administered once daily through oral gavage for 21 consecutive days. On prestudy, 7th, 14th, and 21st days of the experiment, the animals' locomotor activity, muscle grip strength and escape latency time (ELT) were measured. At the end of the study, blood samples were collected from all the animals and they were sacrificed; the organs such as brain, liver, lung, heart, kidney, and stomach were harvested, and absolute organ weight was measured. Blood samples were used for the biochemical analysis. Results: The rats treated with EEST 100, 200, and 400 mg/kg did not show any change in locomotor activity and grip strength and significantly reduced motor resistance. The animals treated with donepezil showed a significant reduction in ELT. The animals treated with EEST 100 and 200 mg/kg showed significant increases in ELT, whereas animals treated with EEST 400 mg/kg did not show significant changes in ELT compared with that of prestudy. At the end of the study, EEST-treated animals did not show significant changes in biochemical parameters, whereas donepezil-treated animals showed a significant increase in cholesterol level compared with control animals. Furthermore, EEST did not affect regular body weight gain, absolute and relative organ weight. Conclusion: EEST did not show neuroprotective effect at lower doses in rats, but the high dose (400 mg/kg) prevented scopolamine-induced memory impairment, and this effect is not significant when compared with standard donepezil.
Keywords: Donepezil, memory, scopolamine, Solanum trilobatum
|How to cite this article:|
Parasuraman S, Hoong SS, Christapher PV, Zou LN, De Wei DL, Loshini S, Ching TH, Leong CH. Effect of ethanolic extract of leaves of Solanum trilobatum on scopolamine-induced memory impairment in Sprague Dawley rats. J Pharm Negative Results 2019;10:41-6
|How to cite this URL:|
Parasuraman S, Hoong SS, Christapher PV, Zou LN, De Wei DL, Loshini S, Ching TH, Leong CH. Effect of ethanolic extract of leaves of Solanum trilobatum on scopolamine-induced memory impairment in Sprague Dawley rats. J Pharm Negative Results [serial online] 2019 [cited 2019 Sep 23];10:41-6. Available from: http://www.pnrjournal.com/text.asp?2019/10/1/41/265144
| Introduction|| |
The use of natural products with therapeutic properties dated back to centuries. Mineral, plant, and animal products were the main drug sources for long time. More than 80% of drug sources were purely natural products or were inspired by the molecules derived from natural sources. The large proportion of natural products in drug discovery has stemmed from the diverse structures and the intricate carbon skeletons of natural products. The secondary metabolites from natural sources perceived as having more biological friendliness than totally synthetic molecules.Solanum trilobatum Linn. (Family: Solanaceae) is one of the promising herbs with multiple medicinal properties.
S. trilobatum (climbing brinjal in English) is also called as purple-fruited pea eggplant which is widely distributed in the southern part of India and Thailand. The plant parts such as berries and flowers are traditionally used for the treatment of respiratory illnesses such as common cold, cough, and asthma. Leaves of this plant can be consumed by mildly frying it in the oil or ghee and then it is grinded. Thorns of the plant parts including on leaves must be removed before cooking as they have mild gastrointestinal toxicity. The toxic effect is due to the presence of solanine, an alkaloid present in solanaceous species. The shade-dried leaves can be powdered and stored for prolonged use. Its use as traditional medicine in India and Thailand is reported. Pharmacognostical profile of this plant was studied and well-documented. Neuroprotective effect of leaves of S. trilobatum is not clear. Hence, this study is planned to investigate the effect of ethanolic extract of leaves of S. trilobatum (EEST) on scopolamine-induced memory impairment in Sprague Dawley (SD) rats.
| Materials and Methods|| |
Preparation of Solanum trilobatum extract
The leaves of S. trilobatum (Solanaceae) plant were collected from Vellore (12.92°N 79.13°E, 220 m above the mean sea level), India. The plant leaves were shade dried for 1 week and powdered using an electrical grinder. The powder was weighed and extracted with ethanol using a Soxhlet apparatus at 75°C ± 5°C. When the solvent became clear (approximately after 6–8 cycles), extraction was completed. The extract was concentrated to a dry mass by evaporation under reduced pressure using rotary distillation. The EEST was stored at room temperature until use.
Healthy, adult, male SD rats weighing 170 ± 10 g, obtained from Central Animal House, AIMST University, were housed and maintained in large, spacious polyacrylic cages at an ambient room temperature with 12-h light/12-h dark cycle, with free access to food and water. Animals were acclimatized for a week prior to the experiment. The study protocol was reviewed and approved by the Institutional Animal Ethics Committee (AUHAEC/FOP/2017/07).
Effect of ethanolic extract of leaves of Solanum trilobatum in scopolamine-induced memory impairment in rats
The male SD rats were divided into six groups with six animals in each as follows:
- Group 1 – Normal control
- Group II – Scopolamine (1 mg/kg, intraperitoneal [i.p.])
- Group III – Scopolamine (1 mg/kg, i.p.) + donepezil (3 mg/kg, p.o.)
- Group IV – Scopolamine (1 mg/kg, i.p.) + EEST (100 mg/kg, p.o.)
- Group V – Scopolamine (1 mg/kg, i.p.) + EEST (200 mg/kg, p.o.)
- Group VI – Scopolamine (1 mg/kg, i.p.) + EEST (400 mg/kg, p.o.).
Memory impairment was induced by i.p injection of freshly prepared scopolamine (1 mg/kg body weight) in 0.1 M citrate buffer (pH 4.5) once daily for 21 days. All the investigational drugs were suspended in 0.5% w/v carboxymethyl cellulose and administered after scopolamine administration for 21 days. The scopolamine and donepezil doses were selected based on previously published studies.,
The drugs and extract were administered once daily through oral gavage for 21 consecutive days. Animal behavioral functions were assessed on prestudy, 7th, 14th and 21st day of the experiment and after 24 h of the last treatment. At the end of the study, the animals were anesthetized and blood samples were collected through retro-orbital plexus and serum was separated. The serum sample was stored at −80°C until the estimation of biochemical markers and lipid profile.,
Body weight analysis
Body weight variations were monitored throughout the study for all the experimental animals at regular intervals.
Behavior and muscular activities
Prestudy day, 7th, 14th and 21st day of the study, behavioral (locomotor activity), muscular coordination (rotarod and wire grip test), and memory function (water maze test) were evaluated.
The animal locomotor behavior was monitored using actophotometer, equipped with acrylic cage, and surrounded by a stainless steel frame, with 8 beams of infrared light on both x and y horizontal axes. The number of beam crossovers, i.e., consecutive interruption of one beam followed immediately by interruption of the adjacent beam by the animal, was recorded by the instrument. The individual rat's activity was monitored at room temperature for 10 min. Each animal was treated with the respective drug, and an activity score was recorded after 15 min. Decreased activity score was taken as an index of central nervous system depression.
Hanging wire grip test
The hanging wire grip test was conducted to assess the neuromuscular strength and coordination of movement of an animal prior to water maze test. Two heavy-based retort stands were kept at a distance of 55 cm, and 1-mm thick thread was tied between the poles at a height of 35 cm. A layer of bedding material was placed to prevent injury to the animal when it falls down. During the test, the thread must be tightly attached to the frame to avoid vibration or unwanted displacement of the thread. Animal was placed on the center of the wire, and the latency to falling time was observed.
Muscle coordination test
This test was conducted using rotarod apparatus. Rotarod test was conducted to assess the muscular coordination of animal prior to water maze test. The starting speed of the rod rotation was adjusted to 4 rpm/min then that was accelerated to 20 rpm/min. Rats were pretrained on the rotarod for 1–3 h prior to the testing. Training consisted of placing a rat on the rotating rod and measuring latency to fall, up to a maximum of 120 s. The rats were tested on the rotarod, 30 min after the treatment with donepezil/EEST to determine the effect of drug or extract.
Morris water maze test
Water navigation test is employed to test spatial learning and memory parameters to evaluate the spatial learning and memory functions. The training was conducted for 3 consecutive days, with four consecutive trials/day for each experimental rat at the intertrial interval of 30 min. In prestudy training sessions, if the animals failed to locate the platform within 180 s, they will be excluded from the study.
The Morris water maze (MWM) consists of a round pool, filled with tap water (23°C–26°C), to the depth of 0.3–0.4 m. The pool was divided into four hypothetical quadrants, with an escape platform was placed 1 cm below the water surface at the center. Four different starting points for rats were placed around the perimeter of the pool. The test was performed on the last day of each week. Escape latency time (ELT) to locate the hidden platform in the water maze was noted as an index of learning.
Gross pathology and organ weight analysis
At the end of the experiment, all the experimental animals were sacrificed on the same day under mild ether anesthesia followed by cervical dislocation. The animal was dissected, and the gross pathology of organs was observed. The organs such as brain, liver, lung, heart, kidney, and stomach were harvested; absolute organ weights were measured, and relative organ weight was calculated.
At the end of the experiment, about 2 mL of blood was collected from retro-orbital plexus using a plain glass tube, and the serum was separated by centrifuging at 3000 RPM for 20 min. The serum sample was used for the biochemical estimation (glucose, aspartate aminotransferase [AST], alanine aminotransferase [ALT], alkaline phosphatase [ALP], urea, and creatinine) and lipid profile (total cholesterol, triglycerides, and high-density lipoprotein [HDL] cholesterol). The calculated low-density lipoprotein (LDL) cholesterol, HDL ratio, and very-low-density lipoprotein [VLDL] were determined. The LDL was calculated using Friedewald's formula (LDL = total cholesterol – HDL − [triglycerides/5]); very LDL (VLDL) was calculated using formula LDL/5.
All the data were expressed as the mean ± standard error of the mean, and the statistical significance between the groups was tested using one-way analysis of variance followed by Tukey's post hoc test. P < 0.05 was considered statistically significant.
| Results|| |
Influence of ethanolic extract of leaves of Solanum trilobatum on body weight variations
EEST and donepezil did not show any significant change in body weight gain in scopolamine-treated animals.
Effect of ethanolic extract of leaves of Solanum trilobatum on behavior and muscular functions
At the end of the study, scopolamine-treated animals showed a reduction in motor resistance. The rats treated with EEST 100, 200, and 400 mg/kg did not show any change in locomotor activity [Figure 1] and grip strength [Figure 2]. EEST showed a significant reduction in motor resistance (in rotarod experiment) at the end of the study when compared with the control group [Figure 3]. However, the donepezil prevented scopolamine-induced impairment in muscular coordination in rats [Figure 3].
|Figure 1: Effect of ethanolic extract of leaves of Solanum trilobatum on locomotor action in scopolamine-treated rats. All the values are mean ± standard error of the mean (n = 6)|
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|Figure 2: Effects of the ethanolic extract of leaves of Solanum trilobatum on hanging wire grip in the scopolamine-treated rats. All the values are mean ± standard error of the mean (n = 6)|
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|Figure 3: Effects of the ethanolic extract of leaves of Solanum trilobatum on muscular coordination in the scopolamine-treated rats. All the values are mean ± standard error of the mean (n = 6). bP < 0.01, and cP <0.001 compared with prestudy fall on time. One-way analysis of variance followed by Turkey's post hoc test|
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Effect of ethanolic extract of leaves of Solanum trilobatum on scopolamine-induced memory impairment
The effect of EEST on scopolamine-induced memory impairment in rats in the MWM test is summarized in [Figure 4]. In the control groups, a slight decrease in ELT was observed; however, administration of scopolamine in the scopolamine control group (Group II) caused memory impairment as there was a significant change in the ELT on the 7th, 14th, and 22nd days. The donepezil treated group showed a significant decrease in ELT, whereas EEST 100 and 200 mg/kg treated animals showed significant increase in ELT on the 7th, 14th, and 22nd days when compared to the ELT of prestudy. At 400 mg/kg, EEST did not show any significant change in ELT on the 7th, 14th, and 22nd days when compared to that of prestudy.
|Figure 4: Effects of the ethanolic extract of leaves of Solanum trilobatum on escape latency time in the scopolamine-treated rats. All the values are mean ± standard error of the mean (n = 6). aP < 0.05, bP < 0.01, and cP < 0.001 compared with prestudy escape latency time. One-way analysis of variance followed by Turkey's post hoc test|
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Effect of ethanolic extract of leaves of Solanum trilobatum on absolute and relative organ weight
It was observed that scopolamine and EEST did not show any significant variations in absolute and relative organ weights.
Effect of ethanolic extract of leaves of Solanum trilobatum on biochemical parameters
There were no significant changes in the biochemical parameters observed in control, scopolamine-treated, and scopolamine + EEST-treated animals, whereas the donepezil-treated animals showed a significantly increased level of total cholesterol, LDL, and VLDL [Table 1] and [Table 2].
|Table 1: Effect of ethanolic extract of leaves of Solanum trilobatum on biochemical parameters of Sprague Dawley rats|
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|Table 2: Effect of ethanolic extract of leaves of Solanum trilobatum on lipid profile of Sprague Dawley rats|
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| Discussion|| |
In this study, the effect of EEST on scopolamine-induced behavioral and memory impairment in rats was investigated. Scopolamine, a muscarinic antagonist, interferes with both short- and long-term memories. Scopolamine induces neuronal injury and causes learning and memory impairment in experimental animals by blocking the muscarinic receptor and increasing the activity of acetylcholinesterase, which was consistent with previous observations.,,, Scopolamine also increases the number of apoptotic neurons in all area of the hippocampus.
Donepezil, a selective acetylcholinesterase inhibitor, was used as a therapy for Alzheimer's disease and also recommended for the enhancement of attention and memory. Donepezil is used as a positive control in this study, and it prevented scopolamine-induced behavioral and memory impairment in rats. In preclinical studies, donepezil improved spatial learning memory in rodents.,
Memory impairment is a neurodegenerative disorder associated with reduced cognitive abilities. The parasympathetic neuronal system plays an important role in cognitive deficits. Anticholinergic drugs such as scopolamine induce a transient disruption of memory in experimental animals and humans by blocking postsynaptic muscarinic receptors. In this study, EEST did not attenuate scopolamine-induced memory impairment at 100 and 200 mg/kg in rats. In a preclinical study, the aqueous extract of leaves of S. trilobatum showed neuroprotective effect against lead acetate-induced neurotoxicity in albino mice. In another study, the extracts of roots and leaves of S. trilobatum showed a significant anti-inflammatory activity which may be due to the presence of sterol or triterpenoid saponins., Chronic administration of nonsteroidal anti-inflammatory drugs may offer some protection against Alzheimer's disease. EEST at 400 mg/kg treated animals did not affect the mean ELT when compared to prestudy ELT, and this may be due to its anti-inflammatory properties. In the computer-aided prediction of biological activities of phytocompounds of S. trilobatum indicated that solanine and beta-solamarine has dementia preventing effect.
In general, scopolamine found to increase locomotor activity due to scopolamine-induced hyperactivity., In contrary, studies also demonstrated decreased locomotor activity and muscular strength in scopolamine-treated animals., A few other studies reported that that scopolamine did not influence locomotor activity in rodents., This study concurs the noninfluence of scopolamine in locomotor activity.
The rotarod test was used to assess motor activity coordination and muscle strength in experimental animals. In the rotarod experiment, scopolamine-treated and scopolamine + EEST-treated animals showed significant decreases in motor resistance. The reduction in motor resistance indicates the development of apraxia in animals treated with scopolamine. This may be due to the increases in the levels of acetylcholinesterase and malondialdehyde in the cortex and hippocampus of the brain. Both cerebral cortex and hippocampus are linked to motor and cognitive functions. These areas are sensitive to oxidative stress, and increased oxidative stress may reduce motor and cognitive performances. In wire grip experiment, scopolamine-treated and scopolamine + EEST-treated animals did not show any change in muscular strength.
Donepezil-treated animals showed elevated levels of total cholesterol, LDL, and VLDL levels and HDL ratio. In clinical studies, increased levels of cholesterol, triglycerides, cholesterol, LDL, and VLDL have been found during donepezil treatment.,,
The results from this study indicate that EEST has no significant neuroprotective effect in scopolamine-induced memory impairment in SD rats. The normal liver and kidney functions were not affected by EEST at any of the three doses treated.
| Conclusion|| |
The effect of EEST on scopolamine-induced memory impairment in SD rats was studied. The study revealed that the EEST has neuroprotective activity only at high dose (400 mg/kg), and this effect is not significant when compared to the standard drug donepezil. A detailed study including different memory testing models is suggested to confirm the memory-enhancing activity and elucidate the possible mechanism of action of memory enhancement.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]