In vivo investigation of the neuroprotective property of Convolvulus pluricaulis in scopolamine-induced cognitive impairments in Wistar rats.

AIM: To investigate the neuroprotective effect of Convolvulus pluricaulis aqueous extract (AE) against scopolamine (1 mg/kg body weight (bwt))-induced neurotoxicity in the cerebral cortex of male Wistar rats.
MATERIALS AND METHODS: The study was carried out on male Wistar rats (age matched, weight 250 ± 20 g). The present study investigated cognitive-enhancing property of AE using Elevated plus maze (EPM) (transfer latency [TL]) and Morris water maze (MWM). Besides evaluating the effect of extract on neurochemical enzymes, in vivo antioxidant and free radical scavenging activities were also screened. All the measured parameters were compared with rivastigmine tartrate (1 mg/kg bwt) which was taken as standard.
RESULTS: Pretreatment of rats with AE (150 mg/kg bwt) significantly reduced scopolamine-induced increase in the TL in EPM, whereas in MWM, administration of extract improved the impairment of spatial memory induced by scopolamine. The activity of acetylcholinesterase (AChE) was significantly inhibited by extract within the cortex and hippocampus. Reduced activities or contents of glutathione reductase, superoxide dismutase, and reduced glutathione within the cortex and hippocampus induced by scopolamine were elevated by the extract. Taken together, it could be postulated that extract may exert its potent-enhancing activity through both anti-AChE and antioxidant action.
CONCLUSION: AE possesses neuroprotective potential, thus validating its use in alleviating toxic effects of scopolamine.

Introduction

Convolvulus pluricaulis (also called Shankhpushpi in Hindi) is a herb that has been extensively investigated for its pharmacological and therapeutic effects. The plant contains alkaloid (Shankhapushpine), volatile oil, flavonoids (kampferol derivatives), phytosterol (beta-sitosterol), carbohydrates (glucose, rhamnose, and starch), ceryl alcohol, and scopoletin.[1] The chloroform fraction of this plant has been found to contain 20-oxodotriacontanol, tetratriacontanoic acid, and 29-oxodotriacontanol. These components were proved to be potent insect antifeedant constituents.[2] Dietary feeding of this plant has been found to increase protein synthesis in the hippocampus, thus enhancing memory and learning in experimental animals.[3] The plant is mainly used as a rasayana, which is advocated for use in rejuvenation therapy. C. pluricaulis has been found to augment both cognitive function and memory-enhancing effects in many behavioral studies.[4] Studies have also showed that the ethanolic plant extract of C. pluricaulis reduced the increased levels of malondialdehyde (MDA) and protein carbonyl. Studies have shown beneficial effect of extract on decreased glutathione peroxidase (GPx) and reduced glutathione (GSH) in hippocampus.[5]

Alzheimer's disease (AD) is the most common cause of dementia in the aged population. AD is characterized by the deposition of the senile plaques mainly composed of β-amyloid (Ab) fragment and neurofibrillary tangles. Despite advances in research, available therapeutic options are limited, thus increasing demand for new drugs. Enhancement of cholinergic neurotransmission represents an important target for treating adult-onset cognitive disorders. Rivastigmine tartrate is a reversible cholinesterase inhibitor. Several studies have shown that the drug produced a dose-related effect on cognitive function that was correlated with the degree of (AChE) inhibition in the cerebrospinal fluid.[6] Hence, the authors have examined the protective effect of C. pluricaulis extract on the impairment of cholinergic and antioxidant activity induced by scopolamine in the brain of male Wistar rats. Histopathological studies were also undertaken to determine the morphological alteration in cerebral cortex.

Materials and Methods

Plant Material

C. pluricaulis (family Convolvulaceae) commonly known as Shankhpushpi were obtained from herbal market in New Delhi. The plant material was identified and authenticated from National Institute of Sciences Communication and Information Resources (NISCAIR) (Ref: NISCAIR/RHMD/Consult 06/77/4191).

Preparation of Aqueous Extract

Roots of the plant C. pluricaulis (200 gm) were triturated in a blender until a finely granulated powder was obtained. The aqueous extract (AE) was obtained from this powder by adding distilled water and soaking it overnight. After filtration, the extract was lyophilized and stored at 4°C for later use. The yield of AE was 17.2 g (8.6%, w/w).

Animals

Adult male rats (Wistar strain) weighing 250 ± 20 g were obtained from central animal facility of Dr. B. R. Ambedkar Center for Biomedical research, New Delhi. They were housed in standard rat cages and maintained on standard diet and water ad libitum. The animal study was performed in accordance with the guidelines provided by the Institutional Animal Ethics Committee (IAEC) of Dr. B. R. Ambedkar Center for Biomedical Research.

Elevated Plus Maze

Rats were divided into six groups of six animals each as follows: Group I animals served as control and received distilled water (10 ml/kg, p.o); Group II animals received scopolamine 30 minutes before Elevated plus maze (EPM) test; and Groups III, IV, and V were fed orally with AE (100, 150, and 250 mg/kg). The treatments were carried out for 7 days. On 7th day, after 90 minutes of administrations, scopolamine (1 mg/kg, i.p.) was injected to Groups II to VI. Transfer latency (TL) was recorded after 30 minutes of injection. Retention was examined after 24 hours. The EPM apparatus consists of two open and two closed arms facing each other and elevated at a height of 50 cm from the ground. Animals were placed individually after oral administration of either vehicle or test drug at the end of either of the open arms and the time taken by the animal to move from open to closed arm; TL was noted on the first day. The time elapsed between the time that the animal was placed on the open arm and the time at which all four legs were inside the enclosed arms was noted as TL. The TL was again recorded 24 hours after the first exposure.

Morris Water Maze

Rats were grouped as six animals each in four groups as follows: Group I animals served as control and received vehicle only; Group II animals received scopolamine (1 mg/kg); Groups III received rivastigmine; and Group IV received AE in doses of 150 mg/kg orally. Scopolamine (1 mg/kg, i.p.) was injected before training. Escape latency and swimming distance were recorded 30 minutes after the administration of scopolamine. The Morris water maze (MWM) test was performed by the method of Morris et al.[7] The apparatus used is a circular water tank (100 cm in diameter) filled to a depth of 30 cm with water (25°C). Four points equally distributed along the perimeter of the tank served as starting locations. The tank was divided arbitrarily into four equal quadrants and a small platform (5 cm width) was located in the center of one of the quadrants. The platform remained in the same position during the training days. The rats were released into the water and allowed 90 seconds to find the platform. Each rat received four trials per day with 5 minutes intertrial interval for 4 days. Escape latency and swimming paths were recorded.

Surgical Manipulation

After retrieval testing, animals were decapitated and brains were immediately dissected out and washed thoroughly with ice-cold saline to remove blood and kept at –80°C. Cerebral cortex and hippocampus were separated following stereotaxic atlas of Paxinos and Watson.[8] Each region was weighed and 10% homogenate (w/v) was made, which was centrifuged at 800 g for 5 minutes at 4°C in an IEC020 refrigerated centrifuge (Rotor No. 894), followed by again centrifugation at 12 365 g for 15 minutes at 4°C to obtain postmitochondrial supernatant which was used for biochemical estimations. For measurement of superoxide dismutase (SOD) activity, tissues were homogenized in 25 mM triethanolamine-diethanolamine buffer (TDB), pH 7.4 and then debris was removed by centrifugation at 32 000 rpm for 1 hour at 4°C.

Biochemical Estimation

The levels of AChE were determined by the method of Ellman et al.,[9] modified by Rocha et al.'s methods.[10] The method is based on the formation of a yellow anion. The method is based on the formation of the yellow anion, 5,5’-dithiobis-2-nitrobenzoic acid (DTNB), measured by absorbance at 412 nm during 2-minute incubation at 25°C. The reaction was initiated by adding 0.8 mM acetylthiocholine iodide. The enzyme activity was expressed in mmolesAcSCh/h/mg of protein. Monoamine oxidase (MAO) levels were measured by the method of Tabor et al.,[11] this method is based on measurement of benzaldehyde formed with benzyl-amine hydrochloride (0.1 M) used as substrate. The absorbance was read at 340 nm and expressed as nmol benzaldehyde/min/mg protein. Lipid peroxidation (LPO) by Okhawa et al.,[12] protein carbonyl by Reznick and Packer,[13] antioxidant enzymes such as GPx by Flohι and Gόnzler,[14] and glutathione reductase (GR) activity was determined according to the method of Carlberg and Mannerviek.[15] GSH content of tissue was determined by the method of Jollow et al.,[16] method is based on the formation of a relatively stable chromophoric product (A412 nm) on reacting a sulfhydryl compound with Ellman's reagent. The quantity of GSH in tissue sample was calculated using standard GSH and values were represented as μg/g protein. SOD activity was measured following the method of Paoletti and Mocali,[17] which involves inhibition of NAD(P)H oxidation mediated by superoxide radical in 0.1 M TDB (pH 7.4). It consists of a purely chemical reaction sequence which generates superoxide from molecular oxygen in the presence of ethylenediamine tetra acetic acid (EDTA), Manganese (II) Chloride (MnCl2) and mercaptoethanol. SOD brings about the inhibition of nucleotide oxidation. Protein estimation was performed using Lowry's method.[18]

Statistical Analysis

Data for the MWM, EPM (TL), and biochemical estimations were expressed as mean ± S.E.M. Difference between the groups and treatments was measured by one-way and two-way analysis of variance (ANOVA). The results were considered to be statistically significant if the probability was 0.05 or less.

Results

Effect on Transfer Latency (Using Elevated Plus Maze)

TL of first day reflected learning behavior of animals, whereas TL of second day reflected retention of information or memory. Scopolamine (1 mg/kg) impaired learning significantly (P < 0.05) as indicated by increased TL. AE (100 mg/kg) administered for 7 days orally showed insignificant effect on TL of first day of training and on second day, whereas AE (150 mg/kg) significantly decreased TL on first day as well as on second day, indicating significant (P < 0.05) improvement of learning and memory. A significant increase (P < 0.05) in the TL was observed in rats administered with AE (250 mg/kg), indicating significant impairment in learning compared with control. Thus, oral administration of extract (150 mg/kg) for 7 days protected the animals from scopolamine-induced impairment in learning and memory (P < 0.01) [Table 1].

Table 1

Effect of Convolvulus pluricaulis AE on elevated plus maze (transfer latency)

Effect of Aqueous Extract on Spatial Memory in Morris Water Maze Test

On the basis of the results obtained from the EPM test, the efficacy of extract (150 mg/kg) in enhancing cognition after the impairment of spatial memory through scopolamine was further evaluated. Data showed that scopolamine increased swimming distance as compared with the control group in all days of evaluation (P < 0.01). AE-treated rats showed a significant (P < 0.05) reduction in swimming distance from day 2 to day 4. Similar reduction in swimming distance was also observed in rats treated with rivastigmine (P < 0.05) [Figure 1a].

Figure 1

Effect of AE (150 mg/kg) and Rivastigmine tartrate (1 mg/kg) on scopolamine (1 mg/kg) induced amnesia measured by hidden-platform acquisition training in Morris water maze test. (a) Swimming distance (b) latency. All values are expressed as Mean ± S.E.M

Animals treated with scopolamine exhibited a longer latency period throughout the training period as compared with the control rats (P < 0.5). Amnesia induced by scopolamine was significantly (P < 0.5) mitigated by AE and the escape latencies reduced from 98.41 ± 10.44 on the first day to 45.66 ± 8.69 on the fourth day. The positive control rivastigmine tartrate (1 mg/kg) antagonized the effect of scopolamine, significantly (P < 0.05) reducing escape latency from 101.11 ± 10.26 on the first day to 87.66 ± 9.14 as compared with scopolamine-treated rats [Figure 1b].

Effect of Aqueous Extract on Probe Trial testing in Morris Water Maze Test

The probe trail testing was performed on the fifth day by removing the platform and allowing each rat to swim freely for 120 seconds. The time and the distance of swimming for each rat spent in the target quadrant (the northeast quadrant, where the platform was removed) were recorded. Compared with scopolamine-treated group, rats treated with AE (150 mg/kg) and rivastigmine tartrate (1 mg/kg) spent more time (P < 0.05) and distance (P < 0.01, P < 0.05) in the target quadrant [Figure 2a and b].

Figure 2

Effect of AE (150 mg/kg) and Rivastigmine tartrate (1 mg/kg) on scopolamine (1 mg/kg)-induced memory defi cit was studied by Probe trial (a) Comparison of the percent of the distance for each rat spent in the target quadrant for probe trial within 120, (b) Comparison of the time of swimming for each rat spent in the target quadrant for probe trial within 120 seconds. All values are expressed as Mean ± S.E.M

Effect of Aqueous Extract on Acetylcholinesterase and Monoamine Oxidase Activity

Scopolamine induced an increase of ~49% and ~1-fold in the enzymatic activity of AChE in brain regions as compared with control rats. AE (150 mg/kg) attenuated the increased activity of AChE in cerebral cortex by ~46% and in hippocampus by ~56% as compared with amnesic group. Administration of rivastigmine tartrate (1 mg/kg, p.o) also inhibited elevated AChE activity by ~24% in cerebral cortex and ~30% in hippocampus, in comparison with amnesic rats. No significant increase in the MAO levels was registered among these animals by scopolamine administration [Table 2].

Table 2

Effect of Convolvulus pluricaulis AE on acetylcholinesterase and monoamine oxidase activity

Effect of Aqueous Extract on Lipid Peroxidation and Protein Oxidation

MDA was increased by ~3-fold and protein carbonyl levels by ~92% in cerebral cortex and ~2-fold in hippocampus by scopolamine as compared with control. AE treatment lowered MDA levels by ~68% and protein carbonyl levels by ~30% and ~61% in brain regions as compared with scopolamine-treated group. Rivastigmine tartrate also attenuated scopolamine-induced LPO by ~51% in Cerebral Cortex, ~43% in hippocampus, and protein carbonyl levels by ~16% and ~44%, respectively, in brain regions [Figure 3a and b.]

Figure 3

Effect of AE (150 mg/kg) and Rivastigmine tartrate (1 mg/kg) on the levels of (a) Lipid peroxidation and (b) Protein oxidation in cerebral cortex and hippocampus of the rat treated with scopolamine. All values are expressed as Mean ± S.E.M. value (n = 6)

Effect of Aqueous Extract on Antioxidant Activity

Scopolamine inhibited GR activity in cerebral cortex and hippocampus by ~50% and ~41%, respectively. This was followed by reduction in SOD activity by ~21% and ~36% in brain regions, while activity of GPx was virtually unchanged. GSH was depleted by ~23% and ~38% in brain regions of amnesic rats as compared with control. Administration of AE restored the activity of GR by ~1-fold and ~51%, whereas SOD registered an increase of ~64% and ~2-fold in brain regions as compared with amnesic group. AE treatment also restored GSH content in cerebral cortex and hippocampus by ~31% and ~50%, respectively, as compared with amnesic group [Table 3].

Table 3

Effect of Convolvulus pluricaulis AE on the antioxidant status within the cortex and hippocampus

Discussion

Scopolamine interferes with memory and cognitive function in human beings and experimental animals by blocking muscarinic receptors.[19] This animal model has been extensively used in research to screen for drugs with potential therapeutic value in dementia.[2021] The EPM paradigmn (TL) was employed in order to deduce the most effective dose among the three doses of the extract, viz. 100, 150, and 250 mg/kg. It was found that dose of 150 mg/kg administered orally for 7 days attenuated the scopolamine-induced learning and memory dysfunction of rats significantly. This dose was further srctinized for neuroprotective properties. In MWM, rats treated with scopolamine showed larger swimming distance and escape latency than rats from control group. These results are in accordance with those described in previous studies.[2223] AE (150 mg/kg) showed inhibitory effect against scopolamine-induced memory impairment in the MWM by reducing total swimming distance and escape latency compared with amnesic group. It is important to notice that the water Morris maze investigated spatial learning and memory, and it is especially sensitive to impaired cholinergic hippocampal function,[24] hence suggesting attenuation of scopolamine-induced spatial learning and memory deficit in male rats by AE. Cholinergic transmission is terminated mainly by acetylcholine hydrolysis through the enzyme AChE, hence essential in maintaining the normal function of the nervous system. Present study showed that increased AChE activity in cortex and hippocampus induced by scopolamine were significantly decreased by AE, hence depicting a relation between the inhibitory effect on AChE activity of AE and learning and memory improvement in male rats. Scopolamine-induced increase in the enzymatic activity of AChE was also reduced by rivastigmine. The primary role of MAO lies in the metabolism of monoamines and in the regulation of monoamine neurotransmitter levels in brain and in the systemic circulation.[25] Results from the present study show that AE did not alter MAO activity, thus suggesting that the neuroprotective effect of AE do not result from direct MAO inhibition. Despite AE not modifying MAO activity directly, an effect on monoamine neurotransmitter should not be ruled out. For instance, it was suggested that P10358 (an acetylcholinesterase inhibitor) alters striatal dopamine metabolism as a direct consequence of cholinergic stimulation and does not interfere with MAO activity or possess dopamine depleting properties.[26] Similar effects were observed with rivastigmine.

Scopolamine administration resulted in a significant increase in lipid damage which was measured in MDA. This was also accompanied by a significant increase in protein carbonyl content, a marker of protein oxidation and an index of oxidative stress. Inhibition of MDA and protein carbonyl by AE suggests its neuroprotective properties. As oxidative damages are mediated by free radicals, it was necessary to investigate the status of endogenous antioxidant enzymes under different treatment conditions. Natural antioxidant enzymes like SOD, GPx, GST, and GSH present the first line of defense against free radical damage under oxidative stress conditions. Scopolamine induced a significant decline in the enzymatic activity of antioxidant enzymes like GR and SOD by scopolamine, whereas GPx activity was little changed. AE (150 mg/kg) and rivastigmine tartrate (1 mg/kg) prevented the decrease in the activity of GR, whereas reduced SOD activity induced by scopolamine was not only preserved, but also elevated higher than that of normal control rats. Studies have reported significantly lower SOD activity than that of non-demented control in the cerebellum, frontal cortex, and hippocampus.[27] Intracellular GSH status is a sensitive indicator of the health of a cell or tissue. Our results showed that GSH levels decreased after scopolamine treatment, and this may lead to elevated levels of lipid and protein oxidative products. Depletion of brain GSH associated with scopolamine treatment was restored significantly by AE. Thus, it can be postulated that AE scavenges ROS and exerts a protective effect against oxidative damage induced by scopolamine by maintaining the activities of glutathione reductase and SOD. The cognitive-enhancing activities of C. pluricaulis might result, in part, from the inhibition of AChE activity, and the reduction in ROS by recovering the antioxidative defense system.

In traditional oriental medicine, it is conventional to use herbs in order to achieve a variety of treatment purposes simultaneously, or to enhance a single effect without causing severe side effects. Hence, C. pluricaulis could be considered as a therapeutic agent to prevent or slow down the development of neurodegenerative diseases such as AD at an early stage.