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Pharmacological actions of Thespesia populnea relevant to Alzheimer's disease.

Abstract

Thespesia populnea (Malvaceae) is a large tree found in the tropical regions and coastal forests of India. Various parts of T. populnea are found to possess useful medicinal properties, such as antifertility, antibacterial, anti-inflammatory, antioxidant, purgative and hepatoprotective activity. The present study was undertaken to investigate the effects of T. populnea bark on cognitive functions, total cholesterol levels and cholinesterase activity in mice. A total of 312 mice divided into 52 different groups were employed in the present investigation. The ethanolic extract of T. populnea (TPE) was administered orally in three doses (100, 200 and 400 mg/kg) for 7 successive days to different groups of young and aged mice. The learning and memory parameters were assessed using elevated plus maze and passive avoidance apparatus. TPE (200 and 400 mg/kg, p.o.) showed significant improvement in memory of young and aged mice. TPE also reversed the amnesia induced by scopolamine (0.4 mg/kg, i.p.) and diazepam (1 mg/kg, i.p.). Furthermore, TPE reduced significantly the central (brain) cholinesterase activity in mice. TPE exhibited a remarkable cholesterol lowering property comparable to simvastatin (a standard drug) in the present study. Furthermore, we observed that, T. populnea bark possessed a powerful memory enhancing activity in mice. Since diminished cholinergic transmission and increased cholesterol levels appear to be responsible for development of amyloid plaques and dementia in Alzheimer patients, TPE may prove to be a useful medicine on account of its multifarious beneficial effects, such as memory improving property, cholesterol lowering, anticholinesterase and anti-inflammatory activity. Therefore, T. populnea bark appears to be a promising candidate for improving memory and it would be worthwhile to explore the potential of this plant in the management of Alzheimer patients.

[c] 2006 Elsevier GmbH. All rights reserved.

Keywords: Thespesia populnea; Amnesia; Memory; Anti-cholinesterase; Cholesterol

Introduction

There has been a steady rise in the number of patients suffering from Alzheimer's disease (AD) all over the world. Quality of life of senior citizens is adversely affected by dementia caused due to degeneration of the cerebral neurons (Agarwal et al., 2002). Memory forms one of the most complex functions of the brain and ultimately involves multiple neuronal pathways and neurotransmitter systems. Facilitation of cholinergic pathways can be looked upon as an important strategy in improving cognitive and behavioral functions (Parle et al., 2004a). AD is a genetically heterogeneous, progressive and neurodegenerative disorder, which is associated with aphasia, apraxia and agnosia, with loss of memory being the cardinal symptom (Parle et al., 2004b; Dhingra et al., 2005). The main histological features of AD include extra cellular deposition of [beta]-amyloid (A[beta]) plaques and intraneuronal neurofibrillary tangles. Recently, cholesterol levels appear to be intimately associated with development of amyloid plaques in humans (Puglielli et al., 2003). Several studies are pouring in showing a strong connection between high cholesterol and high incidence of AD (Puglielli et al., 2003; Sayre et al., 1997; Refolo et al., 2000; Sparks et al., 2000). Therefore, a new approach aimed at controlling blood cholesterol level is gathering momentum for the management of AD. Presently, the allopathic system of medicine principally relies on nootropic agents, such as piracetam, aniracetam, fosracetam, nefiracetam, etc., and anticholinesterases, such as donepezil, metrifonate, rivastigmine, etc. (Balaraman and Shingala, 2002; Mashkovskii and Glushkov, 2001; Gauthier et al., 2003; Potkin et al., 2001; Ringman and Cummings, 1999; Sramek et al., 2000; Sugimoto et al., 2002). Since the allopathic system of medicine is yet to provide a radical cure for AD, it is worthwhile to look for new directions, which would minimize the memory loss of patients with neuropsychiatric disorders. The utility of traditional medicines may be explored for treating patients with dementia.

Thespesia populnea Soland ex. Correa (Malvaceae) is a large tree found in tropical regions and coastal forests of India. Gossypol was found to be the major component of T. populnea (Akhila and Rani, 1993) producing anti-inflammatory (Benhaim et al., 1994) and antifertility effects in rats (Murthy et al., 1981; Ghosh and Bhattacharya, 2004) as well as in human beings (Qian and Wang, 1984). Four naturally occurring quinines, viz. thespone, mansonone-D, mansonone-H, thespone and thespesone, have also been extracted from heartwood of T. populnea (Johnson et al., 1999). In the indigenous system of medicine, the paste of the fruits, leaves and roots of T. populnea are applied externally for various skin diseases. The leaves are applied locally for their anti-inflammatory effects in swollen joints (Anonymous, 1995). The fruits of the plant are used in Ayurveda for the control of diabetes (Sathyanarayana et al., 2004). The barks and flowers possess astringent, hepatoprotective and antioxidant activity in rats (Shirwaikar et al., 1995; Ilavarasan et al., 2003a, b). Immunohistochemical studies revealed the existence of chronic inflammation in certain regions of the brain in AD patients. Since inflammation can be damaging to the host tissue, anti-inflammatory drugs might be beneficial in controlling the progression of AD (McGeer and McGeer, 1999). In the light of above, the present study was undertaken to investigate the effects of T. populnea bark on cognitive functions, total cholesterol levels and cholinesterase activity in mice.

Materials and methods

Plant material

The fresh barks of T. populnea were collected during the month of June 2004 from Erode situated in the state of Tamil Nadu, India. The plant material was taxonomically identified and authenticated by The Head, Raw Materials, Herbarium and Museum division, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi, India. A voucher specimen (NISCAIR/RHM/535/10) has been deposited at the herbarium of NISCAIR and a sample of the specimen is also preserved at Pharmacology Division of Department of Pharmaceutical Sciences, G.J. University, Hisar, India for ready reference.

Preparation of the extract

The freshly collected barks were dried under shade, sliced into small pieces, pulverized using a mechanical grinder and passed through 40 mach sieve, and preserved in an airtight container for further use. The powdered barks (2.25 kg) were extracted with 95% ethanol using a soxhlet extractor, at room temperature. After exhaustive extraction, the ethanolic extract was filtered and concentrated by distillation process. A brownish-black colored residue was obtained (yield 17.8% w/w), which was kept in a desiccator. This ethanolic extract of T. populnea (TPE) bark was used in further experiments.

Animals

A total of 312 mice were employed in the present study. All the experiments were carried out using male, Swiss Albino mice procured from the disease-free small animal house of CCS Haryana Agricultural University, Hisar, Haryana, India. Young (3-4 months old) mice weighing around 20 g and aged (12-15 months old) mice weighing around 35 g were used in the present study. The animals had free access to food and water, and they were housed in a natural (12 h each) light-dark cycle. Food given to mice consisted of wheat flour kneaded with water and mixed with a small amount of refined vegetable oil. The animals were acclimatized for at least 5 days to the laboratory conditions before behavioral experiments. Experiments were carried out between 0900 and 1800h. The experimental protocol was approved by the Institutional Animal Ethics Committee and the care of laboratory animals was taken as per the guidance of CPCSEA, Ministry of Forests and Environment, Government of India (registration number 0436).

Drugs

The drugs used in this study were obtained from following drug houses. Scopolamine hydrobromide (Sigma-Aldrich, USA), diazepam (Ranbaxy, India), 5,5-dithiobis-2-nitrobenzoic acid (DTNB), acetylcholine iodide, eserine salicylate, sodium dihydrogen phosphate, disodium hydrogen phosphate (Hi Media, India), piracetam (UCB India Ltd., India), metrifonate (Sigma-Aldrich, USA), simvastatin (Krebs Biochemicals and Industries Limited, India) and cholesterol diagnostics kit (Erba Diagnostics, Germany).

Vehicle

Plant extract (TPE) was suspended in 2% w/v gum acacia and administered orally in mice. Scopolamine hydrobromide, diazepam, piracetam and metrifonate were dissolved separately in normal saline and injected i.p. Simvastatin was suspended with 0.5% w/v carbox-ymethylcellulose sodium and given orally. Volume of oral administration and i.p. injection was 1 ml/100 g of mouse.

Acute toxicity studies

Acute oral toxicity studies were performed (Ecobichon, 1997) according to OECD-423 guidelines (acute toxic class method). Swiss mice (n = 3) of either sex selected by random sampling technique were employed in this study. The animals were fasted for 4h with free access to water only. The TPE (suspended with 2% w/v gum acacia) was administered orally at a dose of 5 mg/kg initially and mortality was observed for 3 days. If mortality was observed in 2/3 or 3/3 animals, then the dose administered was considered as toxic dose. However, if the mortality was observed in only one mouse out of three animals then the same dose was repeated again to confirm the toxic effect. If mortality was not observed, the procedure was then repeated with higher doses, such as 50, 300 and 2000mg/kg.

Exteroceptive behavioral models

Elevated plus maze

Elevated plus maze served as the exteroceptive behavioral model to evaluate learning and memory in mice. The procedure, technique and end point for testing learning and memory was followed as per the parameters described by the investigators working in the area of psychopharmacology (Itoh et al., 1990; Reddy and Kulkarni, 1998; Dhingra et al., 2003; Parle and Dhingra, 2003). The elevated plus maze for mice consisted of two open arms (16 x 5 [cm.sup.2]) and two covered arms (16 x 5 x 12 [cm.sup.3]) extended from a central platform (5 x 5 [cm.sup.2]), and the maze was elevated to a height of 25 cm from the floor (Dhingra et al., 2004). On the first day, each mouse was placed at the end of an open arm, facing away from the central platform. Transfer latency (TL) was defined as the time taken by the animal to move from the open arm into one of the covered arms with all its four legs. TL was recorded on the first day for each animal. The mouse was allowed to explore the maze for another 2 min and then returned to its home cage. Retention of this learned-task was examined 24 h after the first day trial.

Passive avoidance paradigm

Passive Avoidance Behavior based on negative reinforcement was used to examine the long-term memory (Sharma and Kulkarni, 1990; Parle et al., 2004c). The apparatus consisted of a box (27 x 27 x 27 [cm.sup.3]) having three walls of wood and one wall of Plexiglas, featuring a grid floor (made up of 3 mm stainless-steel rods set 8 mm apart), with a wooden platform (10 x 7 x 1.7) in the center of the grid floor. The box was illuminated with a 15 W bulb during the experimental period. Electric shock (20V, AC) was delivered to the grid floor (Parle and Singh, 2004). Training was carried out in two similar sessions. Each mouse was gently placed on the wooden platform set in the center of the grid floor. When the mouse stepped-down placing all its paws on the grid floor, shocks were delivered for 15s and the step-down latency (SDL) was recorded. SDL was defined as the time (in seconds) taken by the mouse to step down from the wooden platform to grid floor with all its paws on the grid floor. Animals showing SDL in the range of 2-15 s during the first test were used for the second session and the retention test. The second session was carried out 90 min after the first test. When the animals stepped down before 60s, electric shocks were delivered for 15s. During the second test, animals were removed from shock free zone, if they did not step down for a period of 60 s. Retention was tested after 24 h in a similar manner, except that the electric shocks were not applied to the grid floor observing an upper cut-off time of 300 s (Parle et al., 2005).

Biochemical estimations

Collection of blood and brain samples

The animals were sacrificed by cervical decapitation under light anesthesia on the seventh day 90 min after administration of the last dose of TPE. Immediately after decapitation, the trunk blood was collected. Then whole brain was carefully removed from the skull. The collected blood was centrifuged at 3000 rpm for 15 min so as to separate serum. The serum was used for estimation of cholesterol levels. For preparation of homogenate, the fresh whole brain was weighed and transferred to a glass homogenizer and homogenized in an ice bath after adding 10 volumes of 0.9% sodium chloride solution. The homogenate was centrifuged at 3000 rpm for 10min and the resultant cloudy supernatant liquid was used for estimation of cholinesterase level.

Estimation of brain cholinesterase

Cholinesterase activity was measured by the method of Ellman et al. (1961) with a slight modification (Voss and Sachsse, 1970). The cloudy supernatant liquid (0.5 ml) was pipetted out into 25 ml volumetric flask and dilution was made with a freshly prepared DTNB solution (10 mg DTNB in 100 ml of Sorenson phosphate buffer, pH 8.0). From the volumetric flask, two 4 ml portions were pipetted out into two test tubes. Into one of the test tubes, two drops of eserine solution was added. Substrate solution (1 ml) (75 mg of acetylcholine iodide per 50 ml of distilled water) was pipetted out into both tubes and incubated for 10min at 30[degrees]C. The solution in the tube containing eserine was used for zeroing the colorimeter. The resulting yellow color is due to reduction of DTNB by certain substances in the brain homogenate and due to non-enzymatic hydrolysis of substrate. After having calibrated the instrument, change in absorbance per minute of the sample was read at 420 nm (Dhingra et al., 2006).

Estimation of serum total cholesterol

CHOD-PAP method (Henry, 1974; Allain et al., 1974) was used for the estimation of serum total cholesterol. In this method, the blank sample, standard sample and test sample were pipetted into the respective reaction vessels using a micro-pipette. For the blank sample, 20 [micro]l distilled water and 1000 [micro]l working reagent were mixed. For the standard sample, 20 [micro]l standard cholesterol and 1000 [micro]l working reagent, while for the test sample, 20 [micro]l serum and 1000 [micro]l working reagent were mixed. These mixtures were incubated for 10min at 37 [degrees]C. The absorbance was read at 510 and 630 nm (Filters 1 and 2) against the blank sample by using autoanalyzer (Erba Mannheim, Chem-5, Plus [V.sub.2]).

Experimental design

A total of 312 mice divided into 52 different groups were employed in the present investigation. Each group comprised of a minimum of six animals.

Group I: Control group for young mice. Vehicle of the extract was administered orally for 7 successive days. TL was recorded after 90 min of vehicle administration on day 7 and retention was examined after 24 h (i.e. on eighth day).

Group II: Positive control for young mice. Piracetam (400mg/kg, i.p.) was injected to young mice for 7 successive days. TL was recorded after 60min of i.p. injection on day 7 and retention was examined after 24 h (i.e. on eighth day).

Groups III, IV and V: TPE (100, 200 and 400mg/kg, respectively) was administered orally for 7 successive days to young mice. TL was noted after 90min of the extract administration on day 7 and after 24 h (i.e. on eighth day).

Group VI: Scopolamine (0.4mg/kg) was injected intraperitoneally into young mice and TL was recorded 45min after injection. Retention was examined after 24 h (i.e. on eighth day).

Group VII: Piracetam (400 mg/kg, i.p) was injected to young mice for 7 successive days. At 60min after the injection of piracetam on the seventh day, scopolamine (0.4mg/kg, i.p.) was injected. TL was noted after 45min of injection of scopolamine, and retention was examined after 24 h (i.e. on eighth day).

Groups VIII, IX and X: TPE (100, 200 and 400 mg/kg, respectively) was administered for 7 successive days orally. Scopolamine (0.4mg/kg) was injected intraperitoneally to young mice at 90 min after administration of extract on day 7. TL was recorded 45 min after injection and after 24 h (i.e. on eighth day).

Group XI: Diazepam (1 mg/kg) was injected intraperitoneally into young mice, and TL was recorded 45 min after injection. Retention was examined after 24 h (i.e. on eighth day).

Group XII: Piracetam (400 mg/kg, i.p) was injected to young mice for 7 successive days. At 60 min after the injection of piracetam on the seventh day, diazepam (1 mg/kg) was injected. TL was noted after 45 min of injection of diazepam (1 mg/kg) and retention was examined after 24 h (i.e. on eighth day).

Groups XIII, XIV and XV: TPE (100, 200 and 400 mg/kg, respectively) was administered for 7 successive days orally. Diazepam (1 mg/kg) was injected intraperitoneally 90 min after administration of extract on day 7. TL was recorded 45 min after injection and after 24 h (i.e. on eighth day).

Group XVI: Control group for aged mice. The vehicle was administered orally for 7 successive days. TL was recorded after 90 min of vehicle administration on day 7 and retention was examined after24h (i.e. on eighth day).

Group XVII: Positive control for aged mice. Piracetam (400 mg/kg, i.p.) was injected to aged mice for 7 successive days. TL was recorded after 60 min of i.p. injection on day 7 and retention was examined after 24 h (i.e. on eighth day).

Groups XVIII, XIX and XX: TPE (100, 200 and 400 mg/kg, respectively) was administered orally for 7 successive days to aged mice. TL was noted after 90 min of the extract administration on day 7 and after24 h (i.e. on eighth day).

Group XXI: Control group for young mice. The vehicle was administered orally for 7 successive days. Shock was delivered for 15 s after 90min of vehicle administration on day 7, and SDL was recorded after 24 h (i.e. on eighth day).

Group XXII: Positive control for young mice. Piracetam (400 mg/kg, i.p.) was injected to young mice for 7 successive days. Shock was delivered for 15 s after 60 min of i.p. injection on day 7, and SDL was examined after 24 h (i.e. on eighth day).

Groups XXIII, XXIV and XXV: TPE (100, 200 and 400 mg/kg, respectively) was administered orally for 7 successive days to young mice. Shock was delivered for 15 s after 90 min of the extract administration on day 7, and SDL was noted after 24 h (i.e. on eighth day).

Group XXVI: Scopolamine (0.4 mg/kg) was injected intraperitoneally into young mice and shock was delivered for 15 s after 45 min of injection and SDL was noted after 24 h (i.e. on eighth day).

Group XXVII: Piracetam (400 mg/kg, i.p) was injected to young mice for 7 successive days. At 60 min after the injection of piracetam on the seventh day, scopolamine (0.4 mg/kg, i.p.) was injected. Shock was delivered for 15 s after 45 min of injection of scopolamine and SDL was examined after 24 h (i.e. on eighth day).

Groups XXVIII, XXIX and XXX: TPE (100, 200 and 400 mg/kg, respectively) was administered orally for 7 successive days to young mice. Scopolamine (0.4 mg/kg) was injected intraperitoneally to young mice at 90 min after administration of extract on day 7. Shock was delivered for 15 s after 45 min of injection and SDL was noted after 24 h (i.e. on eighth day).

Group XXXI: Diazepam (1 mg/kg) was injected intraperitoneally into young mice. Shock was delivered for 15 s after 45 min of injection and SDL was noted after 24 h (i.e. on eighth day).

Group XXXII: Piracetam (400 mg/kg, i.p) was injected to young mice for 7 successive days. At 60 min after the injection of piracetam on the seventh day, diazepam (1 mg/kg) was injected. Shock was delivered for 15 s after 45 min of injection of diazepam (1 mg/kg), and SDL was examined after 24 h (i.e. on eighth day).

Groups XXXIII, XXXIV and XXXV: TPE (100, 200 and 400 mg/kg, respectively) was administered orally for 7 successive days to young mice. Diazepam (1 mg/kg) was injected intraperitoneally to young mice at 90 min after administration of extract on day 7. Shock was delivered for 15 s after 45 min of injection and SDL was noted after 24 h (i.e. on eighth day).

Group XXXVI: Control group for aged mice. The vehicle was administered orally for 7 successive days. Shock was delivered for 15 s after 90 min of vehicle administration on day 7, and SDL was examined after 24 h (i.e. on eighth day).

Group XXXVII: Positive control for aged mice. Piracetam (400 mg/kg, i.p.) was injected to aged mice for 7 successive days. Shock was delivered for 15 s after 60 min of i.p. injection on day 7, and SDL was examined after 24 h (i.e. on eighth day).

Groups XXXVIII, XXXIX and XL: TPE (100, 200 and 400 mg/kg, respectively) was administered orally for 7 successive days to aged mice. Shock was delivered for 15 s after 90 min of extract administration on day 7, and SDL was noted after 24 h (i.e. on eighth day).

Group XLI: Control group for young mice. The vehicle was administered orally for 7 successive days. The blood and brain samples were collected for estimation of cholinesterase and total cholesterol levels after 90 min of vehicle administration on day 7.

Group XLII: Control group for aged mice. The vehicle was administered orally for 7 successive days. The blood and brain samples were collected for estimation of cholinesterase and total cholesterol levels after 90 min of vehicle administration on day 7.

Group XLIII: Metrifonate (50 mg/kg, i.p.), an anticholinesterase agent (standard drug), was injected to young mice, 60 min before dissecting the animals for estimation of cholinesterase levels.

Group XLIV: Metrifonate (50 mg/kg, i.p.) was injected to aged mice, 60 min before dissecting the animals for estimation of cholinesterase levels.

Group XLV: Simvastatin (5 mg/kg), a cholesterol lowering agent (standard drug), was given orally to young mice for 7 successive days. The animals were dissected for estimation of total cholesterol levels after 90 min of drug administration on day 7.

Group XLVI: Simvastatin (5 mg/kg) was given orally to aged mice for 7 successive days. The animals were dissected for estimation of total cholesterol levels after 90 min of drug administration on day 7.

Groups XLVII, XLVIII and XLIX: TPE (100, 200 and 400 mg/kg) was given orally to young mice for 7 successive days. The animals were dissected for estimation of cholinesterase and total cholesterol levels after 90 min of extracts administration on day 7.

Groups XLX, XLXI and XLXII: TPE (100, 200 and 400 mg/kg) was given orally to aged mice for 7 successive days. The animals were dissected for estimation of cholinesterase and total cholesterol levels after 90 min of extracts administration on day 7.

Statistical analysis

All the results were expressed as mean [+ or -] standard error (SEM). Data were analyzed using one-way ANOVA followed by Dunnett's t-test and Student's unpaired t-test. p-Values < 0.05 were considered as statistically significant.

Results

Acute oral toxicity

T. populnea extract did not produce any mortality even at the highest dose (2000mg/kg, p.o.) employed. All the doses (5, 50 and 300mg/kg, p.o.) of TPE were thus found to be non-toxic. Three doses (100, 200 and 400 mg/kg) of TPE were selected for further psycho-pharmacological and biochemical studies.

Effect on TL (using elevated plus maze)

TL of first day (on seventh day of drug treatment) reflected learning behavior of animals. Whereas, TL of next day reflected retention of information or memory. TPE (100 mg/kg) administered for 7 days orally did not have any significant effect on TL of seventh day and eighth day in elevated plus maze test. The young and aged animals treated orally with 200 and 400 mg/kg showed remarkable reduction (p < 0.001) in TL of seventh day as well as eighth day, indicating significant improvement in learning and memory (Figs. 1 and 2). Scopolamine hydrobromide (0.4 mg/kg, i.p.) and diazepam (1 mg/kg, i.p.) injected before training significantly increased (p < 0.001) TL on days 7 and 8 indicating impairment in learning and memory (Figs. 3 and 4). The TPE at higher dose levels (200 and 400 mg/kg, p.o. for 7 successive days) successfully reversed memory deficits induced by scopolamine (p < 0.001) and diazepam (p < 0.001). Piracetam (used as the positive control) at a dose of 400 mg/kg, i.p. also improved learning and memory in both young and aged mice and reversed the amnesia induced by scopolamine and diazepam as expected.

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Effect on SDL (using passive avoidance paradigm)

SDL of second day/eighth day of drug treatment reflected the long-term memory of animals. TPE (100 mg/kg, p.o.) did not exert any significant effect on SDL (191.2 [+ or -] 9 s) of young mice as compared to control group (170 [+ or -] 7.8 s). On the other hand, the higher doses of 200 and 400 mg/kg of the extract administered orally in young (3-4 months) mice for 7 days markedly (p < 0.001) increased SDL (234.4 [+ or -] 8.2 and 253.2 [+ or -] 4.9, respectively) as compared to the control group (170.8 [+ or -] 7.79). Aged (12-15 months) mice showed significantly (p < 0.001) low SDL (132.2 [+ or -] 5.9) thereby indicating that aging had produced amnesia in these animals. TPE (200 and 400mg/kg, p.o.) successfully reversed (p < 0.001) aging induced amnesia (193.6 [+ or -] 12.3 and 235.8 [+ or -] 6.5). Scopolamine (0.4mg/kg, i.p.) and diazepam (1 mg/kg, i.p.) significantly (p < 0.001) decreased SDL (110.2 [+ or -] 8.8 and 119.2 [+ or -] 7.6) as compared to the control group of young mice, indicating impairment of memory (amnesia). TPE (200 and 400 mg/kg, p.o.) administered for 7 days significantly reversed amnesia induced by both scopolamine (SDL values were 189.4 [+ or -] 8.9 and 237.8 [+ or -] 8) and diazepam (SDL values were 199.6 [+ or -] 8.6 and 232.2 [+ or -] 4.2). The groups of mice, which were treated with piracetam (400 mg/kg, i.p.) for 7 successive days, showed improvement (p < 0.001) in memory of young (SDL was 255.8 [+ or -] 3) as well as aged (SDL was 240.8 [+ or -] 2.8) mice and reversed amnesia induced by scopolamine (SDL was 242.4 [+ or -] 3.5) and diazepam (SDL was 238.4 [+ or -] 4.7).

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Effect on brain cholinesterase activity

The lowest dose of TPE (100 mg/kg, p.o.) did not produce any effect on cholinesterase activity in young and aged mice. However, in higher doses (200 and 400 mg/kg, p.o.), TPE showed a remarkable reduction in brain cholinesterase activity in young and aged mice, as compared to respective control groups by using Ellman's kinetic colorimetric method. The percentage reductions in cholinesterase activity were 12.94% (p < 0.01) at the dose of 200mg/kg and 20.01% (p < 0.001) at the dose of 400 mg/kg in young mice (Fig. 5). Whereas, the inhibition of cholinesterase activity was 13.27% (p < 0.001) at the dose of 200 mg/kg and 22.82% (p < 0.001) at the dose of 400 mg/kg in aged mice (Fig. 6). Metrifonate (50 mg/kg, i.p.), used as a standard drug, showed remarkable reduction of brain cholinesterase activity in young (p < 0.001; 24.90%) and aged (p < 0.001; 26.44%) mice (Figs. 5 and 6).

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Effect on total cholesterol level

The animals receiving TPE (200 and 400 mg/kg, p.o.) for 7 days successively showed significant reduction in total cholesterol levels of young as well as aged mice, when tested using autoanalyzer following colorimetric method. The extent of reduction in total cholesterol levels of young mice were 13.22% (p < 0.05) and 16.55% (p < 0.01) at doses of 200 and 400mg/kg of TPE, respectively (Fig. 7). Similarly, the reductions were 13.18% (p < 0.05) at the dose of 200mg/kg and 22.84% (p < 0.01) at the dose of 400mg/kg in aged mice (Fig. 8). The extent of reductions in total cholesterol levels with simvastatin (a standard cholesterol lowering agent) were 28.21% (p < 0.001) in young animals and 28.56% (p < 0.001) in aged animals (Figs. 7 and 8).

Discussion

AD is a genetically heterogeneous neurodegenerative disorder, which is slow in onset but relentless in progress. It is characterized by aphasia, apraxia and agnosia with loss of memory as the main symptom (Palmer, 2002; Parle et al., 2004b). Despite the severity and high prevalence of this disease, allopathic system of medicine is yet to provide a satisfactory antidote. Therefore, we were motivated to explore the potential of medicinal plants from Himalayan flora to manage this deadly disease (AD). In the present study, T. populnea extract administered orally for 7 days improved the memory of mice as reflected by diminished TL and enhanced SDL values as compared to control animals. Additionally, TPE reduced central cholinesterase activity. Furthermore, pretreatment with TPE for 7 days protected the animals from memory deficits produced by scopolamine and diazepam. These findings suggest the possible neuroprotective role for T. populnea.

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The main histological features of AD include extra-cellular protein deposits termed as A[beta] plaques, A[beta] deposits in blood vessels and intraneuronal neurofibrillary tangles (Sayre et al., 1997; Sparks et al., 1994). Abnormal accumulation of cholesterol levels increase A[beta] in cellular and most animal models of AD; and drugs that inhibit cholesterol synthesis lower A[beta] in these models (Puglielli et al., 2003; Mori et al., 2001). A number of epidemiological studies point out that high level of cholesterol contribute to the pathogenesis of AD (Koudinov and Koudinova, 2001; Jarvik et al., 1995; Kuo et al., 1998; Cho et al., 2003; Fernandes et al., 1999). Interestingly, the animals, which were treated with TPE, showed significant reduction in cholesterol levels in young and aged mice as compared to control group. Therefore, it seems likely that T. populnea may prove to be a useful anti-Alzheimer agent, in view of its memory enhancing property observed in the present study. This suggestion is further substantiated by the reports available in the literature stating that statins, which reduce serum cholesterol levels, protect against dementia and AD (Jick et al., 2000). Researchers have reported that lovastatin and pravastatin reduced the risk of AD up to 73% (Wolozin and Behl, 2000; Fassbender et al., 2001).

It has been observed that elderly patients suffering from AD showed reduction in symptoms of AD upon chronic use of anti-inflammatory drugs (Rao et al., 2002; Stephan et al., 2003). Epidemiological studies have almost confirmed that non-steroidal anti-inflammatory drugs reduce the incidence of AD (Breitner, 1996). T. populnea has been shown to produce anti-inflammatory action in rodents and human neutrophils (Benhaim et al., 1994; Anonymous, 1995). This anti-inflammatory effect of T. populnea would certainly help Alzheimer patients by taking care of the inflammatory component of the AD. Oxygen free radicals are implicated in the process of age-related decline in cognitive performance and may be responsible for the development of AD in elderly persons (Sinclair et al., 1998; Berr, 2002; Butterfield and Lauderback, 2002; Floyd and Hensley, 2002; Perry et al., 2002; Rogers et al., 2003; Bickford et al., 2000). T. populnea has been reported to possess antioxidant property as well (Ilavarasan et al., 2003a). The neuroprotective effect of TPE may be attributed to its antioxidant property by virtue of which susceptible brain cells get exposed to less oxidative stress resulting in reduced brain damage and improved neuronal function.

Acetylcholine is considered as the most important neurotransmitter involved in the regulation of cognitive functions. There is extensive evidence linking the central cholinergic system to memory (Ghelardini et al., 1998; Peng et al., 1997; Olney, 1990; Parle et al., 2004a). Cognitive dysfunction has been shown to be associated with impaired cholinergic function and the facilitation of central cholinergic activity with improved memory (Bhattacharya et al., 1993). Selective loss of cholinergic neurons and decrease in cholinacetyltransferase activity was reported to be a characteristic feature of senile dementia of the Alzheimer's type (Agnolli et al., 1983). Our research findings using Glycyrrhiza glabra, Myristica fragrans and ascorbic acid have displayed a link between memory improving effect and cholinesterase activity (Dhingra et al., 2006). In the present study, the TPE showed elevation of acetylcholine level by significant reduction of cholinesterase activity in brain in treated young and aged mice. Previous pharmacodynamic studies with T. populnea showed that this plant possessed useful anti-inflammatory and antioxidant properties. In the present study, we observed that T. populnea extract (i) lowered serum cholesterol in mice, (ii) inhibited acetylcholinesterase enzyme, thereby elevating acetylcholine concentration in brain homogenate and (iii) ultimately improved memory of both young and aged mice. Thus, a combination of anticholinesterase, anti-inflammatory, antioxidant and cholesterol lowering effects exhibited by T. populnea may all be eventually responsible for the memory improving effect observed in the present study.

Conclusion

In the present study, we observed that Thespesia populnea extract (i) lowered serum cholesterol in mice, (ii) elevated acetylcholine level in brain and (iii) ultimately improved memory of both young and aged mice. In the light of above, it may be worthwhile to explore the potential of this plant in the management of Alzheimer patients.

Acknowledgments

Authors are deeply grateful to Indian Council of Medical Research (ICMR), New Delhi, Government of India for the financial support to this study in the form of SRF. We owe a deep sense of gratitude to Dr. R.P. Bajpai, Hon'ble Vice Chancellor of Guru Jambheshwar University, Hisar for his constant encouragement and inspiration. The authors are thankful to Dr. D.N. Mishra, Chairman, Department of Pharmaceutical Sciences of Guru Jambheshwar University, Hisar for providing infrastructural facilities to carry out this project. We are grateful to Dr. P.K. Kapoor, Scientist In-Charge, disease-free animal house, CCS Haryana Agricultural University, Hisar for continuous supply of animals.

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M. Vasudevan, M. Parle*

Pharmacology Division, Department of Pharmaceutical Sciences. Post Box--38, Guru Jambheshwar University (State Technical University), Hisar-125001, Haryana, India

*Corresponding author. Tel.: +91 09812161998; fax: + 91 1662276240.

E-mail address: mparle@rediffmail.com (M. Parle).
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Author:Vasudevan, M.; Parle, M.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:Nov 1, 2006
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