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Antioxidant property of Celastrus paniculatus Willd.: a possible mechanism in enhancing cognition
Phytomedicine 9: 302–311, 2002 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/phytomed
Phytomedicine
Antioxidant property of Celastrus paniculatus Willd.: a possible mechanism in enhancing cognition M. H. V. Kumar and Y. K. Gupta Neuropharmacology laboratory, Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India
Summary In the present study aqueous, methanolic, chloroform and petroleum ether extracts of seeds of Celastrus paniculatus were investigated for their effect on cognitive functions in rats. Male Wistar rats weighing 200–250 g each were used to study effect on learning and memory through use of the shuttlebox, step-through, step-down and elevated plus maze paradigms. Only the aqueous seed extract (200 mg/kg body wt. for 14 days) showed an improvement in learning and memory in both the shuttle-box and step-through paradigms. Therefore, further experiments were conducted using the aqueous extract at 100, 200 and 300 mg/kg body wt. doses in different paradigms of cognition. All three doses of the aqueous extract increased the number of avoidances in the shuttle-box and step-through latency the in step-through apparatus, but no significant difference was observed between the doses tested. In the step-down apparatus, the 200- and 300-mg/kg body wt. doses of aqueous extract showed a significant increase in step-down latency, whereas no significant difference was observed in the elevatedplus-maze paradigm between drug-treated and vehicle-treated groups. Since the behavioral impairments are associated with oxidative stress, we investigated the effect of the aqueous extract on oxidative stress parameters. Among the three doses tested, only 200 and 300 mg/kg body wt. stimulated a significant decrease in the brain levels of malondialdehyde, with simultaneous significant increases in levels of glutathione and catalase. The present findings indicate that the aqueous extract of Celastrus paniculatus seed has cognitive-enhancing properties and an antioxidant effect might be involved. Key words: Celastrus paniculatus, learning and memory, active avoidance, passive avoidance, antioxidant
j Introduction Ayurveda, the ancient traditional system of medicine has described Celastrus paniculatus Willd. (CP) (Celastraceae) for prevention and treatment of a wide number of disease. According to Ayurveda, CP may be employed as a stimulant nervine tonic, rejuvenant, sedative, tranquilizer and diuretic. It is also used in the treatment of leprosy, leucoderma, rheumatism, gout, paralysis and asthma (Vaidyaratnam, 1994). Sporadic reports in the scientific literature also exist to suggest that the oil of CP and its extracts exhibit the following actions: antiviral (Bhakuni et al., 1969), antibacterial (Patel and Trivedi, 1962), insecticidal (Atal et al.,
1978), analgesic, anti-inflammatory (Ahmad et al., 1994; Dabral and Sharma, 1983), antimalarial (Ayudhaya et al., 1987), antifatigue (Kakrani et al., 1985), antispermatogenic (Wangoo and Bidwai, 1988), hypolipemic (Khanna et al., 1991), sedative (Ahumada et al., 1991) and anticonvulsant (Joglaker and Balwani, 1967). The indigenous people employ CP also as a general cognitive-enhancing agent. Several laboratory reports are now available to support these uses. Karanth et al. (1980), Karanth et al. (1981), and Nalini et al. (1995), reported that rats treated with CP oil improved their performance in the raised-platform shock avoidance 0944-7113/02/09/04-302 $ 15.00/0
Antioxidant property of Celastrus paniculatus Willd. and two-way passive avoidance paradigms. Nalini et al. (1986) also reported that chronic treatment with CP oil produced improvement in I. Q. scores and decreased the content of catecholamine metabolites in mentally retarded children. However, a comparative study of the different extracts of the seed on paradigms of learning and memory is lacking. Therefore, in the present study the aqueous, methanolic, chloroform and petroleum ether extracts of CP seed were evaluated in different learning and memory paradigms in rats. Reactive oxygen species (ROS) from both endogenous and exogenous sources are involved the aging and age-associated neurodegenerative disease, and reports suggest that ROS are associated with cognitive deficits (Good et al., 1996; Gassen and Youdim, 1997). Recently Russo et al. (2001) have reported that CPshows antioxidant effects in in vitro tests. Therefore, the effect of the CPextract which showed the maximum cognitiveenhancing property was also studied in markers of brain oxidative stress, namely, malondialdehyde (MDA), glutathione, superoxide dismutase (SOD) and catalase.
j Materials and Methods Animals
Studies were carried out using male Wistar rats weighing 200–250 g each. They were obtained from the central animal facility of the All India Institute of Medical Sciences, New Delhi, and stock bred in the departmental animal house. The rats were group-housed in polyacrylic cages (38 × 23 × 10 cm) with not more than 4 animals per cage and maintained under standard laboratory conditions (temperature 25 ± 2 °C) with a light and dark cycle (14 ± 1 h: 10 ± 1 h). They were allowed free access to standard dry pellet diet (Golden Feeds, India) and tap water ad libitum. All behavioral tests were performed between 9:00 and 13:00 h. All procedures described were reviewed and approved by the Institutional Committee for Ethical use of Animals. Plant material
Seeds of CP were procured from the local commercial market of New Delhi. The samples were then authenticated for their correct botanical identity by the chief Botanist, Department of Drug Standardization, Dabur Research Foundation, Ghaziabad, India. The whole plant was dried and coarsely ground. For the preparation of the aqueous extract, coarse powder of the seed was extracted with 8 parts boiling water for 5 h and filtered to yield the extract. The extract was then concentrated and finally dried to a powder. For the methanolic, chloroform and petroleum ether extracts, the coarse powder of the seed was extracted with 5 parts methanol,
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chloroform or petroleum ether at 60–65 °C for 5 h and filtered to yield the extracts. The extracts were then concentrated in vacuo and concentration was continued to completely remove the solvent. The percentage (w/w) yields of the aqueous, methanolic chloroform and petroleum ether extracts were 8, 24.8, 25.6 and 20 percent, respectively. Treatment schedules
The animals were divided in two sets of 5 groups to test the 4 extracts (aqueous, methanolic, chloroform and petroleum ether) and the vehicle on the shuttle-box and step-through apparatus paradigms . In both the sets, the 5 groups of animals were administered vehicle orally (Tween 80:water (1:5)), or aqueous extract, methanolic extract, chloroform extract or petroleum ether extract, each at 200 mg/kg body wt.for 14 days. One set was tested on the shuttle-box and the number of avoidances to unconditioned stimulus (US) was noted. Another set was tested on the step-through apparatus and the prolongation of step-through latency was determined. Of the 4 extracts tested, the aqueous extract showed a significant improvement in learning and memory in both the shuttle-box and step-through paradigms. Therefore, further experiments were conducted using the three doses (100, 200 and 300 mg/kg body wt.) of only the aqueous extract in the shuttle-box, step-through apparatus, step-down apparatus and elevated-plus-maze paradigms. The rats were divided into 3 sets of 4 groups each. The 4 groups of all three sets were administered vehicle (Tween 80:Water (1:5)), or aqueous extract at 100, 200 and 300 mg/kg body wt., respectively, for 14 days. The first set of animals was tested on the shuttle-box and the number of avoidances to US was noted. The second set was tested on the step-through paradigm and prolongation of step-through latenc was noted. In the third set of experiments, two memory paradigms were tested. First, in the step-down apparatus to note the step-down latency, followed by the elevated-plus-maze paradigm, to determine the effect on transfer latency. Following the, behavioral test on the fourteeth day, the animals of the third set were sacrificed for estimation of markers of oxidative stress. Behavioral test
• Two-way active avoidance with negative (punishment) reinforcement: Shuttle-box: The apparatus was a box (29 × 23 × 21 cm) divided into two equal compartments with an opening of 8 × 8 cm in middle of the wall separating the 2 compartments. A light source (25 W bulb) and a buzzer (70 db) were fitted in both compartments and switched on alternatively in the two compartments. These were used as conditioned stimulus (CS). The unconditioned stimulus (US) was an electric shock
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(50 V AC) applied to the grid floor for 2 s. Each trial consisted of conditioned stimulus for 10 s, followed by unconditioned stimulus of 2 s electric shock. The intertrial interval was 8 s. Thus, the total time for one trial was 20 s. An avoidance response was recorded when the animal avoided the US (i.e., animals crossed to the other compartment) within 10 s after the onset of CS. The rats were treated orally through an intragastric feeding tube with the drugs for 14 days. On the sixth day each rat was adapted to the shuttle-box. For this purpose, each rat was placed in a compartment of the apparatus and 20 conditioned stimuli (12-s light & buzzer and 8-s interval) were applied without electrical reinforcement. On the seventh day, one h after the administration of the drugs, each rat was trained with 50 such trials called “initial trials”. The avoided response (A) and unavoided response (UA) were recorded. Retention tests were given 24 h, 48 h and 7 days after the initial trial and termed as the first, second and third retention trials (RT), respectively (Vesselin et al., 1993). • Passive avoidance with negative (punishment) reinforcement: Step-through: The apparatus consisted of equal-size light and dark compartments (30 × 20 × 30 cm). A 40-watt bulb was fixed 30 cm above its floor, in the center of the roof of the light compartment. The floor consisted of a metal grid connected to a shock scrambler. The two compartments were separated by a trap door that could be raised to 10 cm. The rats were treated orally through an intragastric feeding tube for 14 days. On the seventh day of drug administration, rats were placed in the light compartment and the time taken to enter the dark compartment, with all four paws inside it, was recorded and termed as initial latency (IL). Immediately after the rat entered the dark chamber, with all four paws inside the dark chamber, the trap door was closed and an electric foot shock (50 V AC) was delivered for 2 s. Rats that had an initial latency of more than 60 s were excluded from experiments. 3 h, 24 h and 7 days later, the latency (time lapse before each animal entered the dark compartment) was recorded in the same manner as in the acquisition trial and were termed as first, second and third retention latencies (RL), respectively, but the foot shock was not delivered, and the latency time was recorded to a maximum of 600 s (Vesselin et al., 1993). • Passive avoidance with negative (punishment) reinforcement: step-down: The apparatus consisted of a chamber (29 × 30 × 29 cm) with a metal grid floor. A wooden platform (10 × 8 cm) was fixed in the center of the grid floor. The rats were treated orally through a probe for 14 days. On the seventh day of drug treatment, the rats were placed on the platform and trained to remain on it for a longer time to escape the electric footshock. Each descent of the animal to the grid floor was punished by electric footshock for 2 s (50 V a.c).
Each training session consisted of 5 trials. Time taken to descent in the last trial (fifth) was considered as “Initial latency” (IL). Retention latencies were tested 3 h, 24 h and 7 days later by placing the animal onto the platform again and measuring the time of descent and were termed as first, second, and third retention latencies (RL), respectively (Vesselin et al., 1993). • Elevated Plus Maze: The plus maze consisted of two opposite open arms (50 × 10 cm), crossed with two closed arms of the same dimensions, with 40-cm high walls. The arms were connected with a central square (10 × 10 cm). On day 7 of drug treatment, rats were placed individually at one end of an open arm, facing away from the central square. The time taken for the rat to move from the open arm and enter into one of the closed arms was recorded and termed as “transfer latency” (TL). Each animal was allowed to explore the maze for 30 sec after recording transfer latency. After 24 h and 7 days, the rat was placed similarly on the open arm and retention latency (RTL) was noted again (Bhattacharya, 1994). While doing the experiments with the shuttle-box, step-through and step-down apparatus, the grid as well and the rat paw were moistened with water before delivering foot shock, as it is known to reduce the wide interanimal variability in paw-skin resistance among rats. Biochemical estimation of markers of oxidative stress
On day 14 following the behavioral testing, animals were decapitated under ether anesthesia and the brains quickly removed, cleaned with ice-cold saline and stored at –80 °C. • Tissue preparation: Brain-tissue samples were thawed and homogenized with 10 times (w/v) homogenizer in ice-cold 0.1 M phosphate buffer (pH 7.4). Aliquots of homogenates from the rat brains were separated and used to measure protein, lipid peroxidation and glutathione. The remaining homogenates were centrifuged at 15,000 rpm for 60 min and the supernatant was then used for enzyme assays. Catalase activity was determined immediately after sample preparation and superoxide dismutase was determined within 24 h. Protein concentration was determined according to Lowry et al. (1951) using purified bovine serum albumin as a standard. • Estimation of Malondialdehyde: Malondialdehyde (MDA), a measure of lipid peroxidation, was estimated as described by Jainkang et al. (1990). The reagents acetic acid 1.5 ml (20 %), pH 3.5, 1.5 ml thiobarbituric acid (0.8 %) and 0.2 ml sodium dodecyl sulphate (8.1 %) were added to 0.1 ml of processed tissue samples, then heated at 100 °C for 60 min. The mixture was cooled under tap water and 5 ml of n-butanol:pyridine (15:1), and 1 ml of distilled water was added. The mixture was vortexed vigorously. After centrifugation at 4000 rpm for 10 min,
Antioxidant property of Celastrus paniculatus Willd. the organic layer was separated and absorbance was measured at 532 nm using a spectrophotometer. The concentration of MDAis expressed as nmol/g tissue. • Estimation of glutathione: Glutathione was measured according to the method of Ellman (1959). An equal quantity of homogenate was mixed with 10 % trichloroacetic acid and centrifuged to separate the proteins. To 0.01 ml of this supernatant, 2 ml of phosphate buffer (pH 8.4), 0.5 ml of 5’5-dithiobis (2-nitrobenzoic acid), and 0.4 ml of double distilled water were added. The mixture was vortexed and the absorbance read at 412 nm within 15 min. The concentration of glutathione was expressed as µg/g tissue. • Estimation of catalase: Catalase activity was measured by the method of Aebi (1974). 0.1 ml of supernatant was added to a cuvette containing 1.9 ml of 50-mM phosphate buffer (pH 7.0). The reaction was started by the addition of 1.0 ml of freshly prepared 30-mM H2O2. The rate of decomposition of H2O2 was measured spectrophotometrically from changes in absorbance at 240 nm. The activity of catalase was expressed as units/mg protein. • Estimation of superoxide dismutase (SOD): The SOD activity of the brain tissue was analyzed by the method described by Kakkar et al. (1984). The assay mixture contained 0.1 ml of sample, 1.2 ml of sodium pyrophosphate buffer (pH 8.3, 0.052 M), 0.1 ml phenazine methosulphate (186 µM), 0.3 ml of 300 µM nitroblue tetrazolium, and 0.2 ml NADH (750 µM). Reaction was started by addition of NADH. After incubation at 30 °C for 90 sec, the reaction was stopped by the addition of 0.1 ml glacial acetic acid. The reaction mixture was stirred vigorously with 4.0 ml of n-bu-
Fig. 1. Effect of aqueous, methanolic, chloroform and petroleum ether extracts of Celastrus paniculatus (200 mg/kg body wt.) on learning and memory in the shuttle-box. On the ordinate; mean number of avoidances (A) per 50 trials ± SEM of eight animals each; on the abscissa: IT- initial trial on day 7 of drug administration, 1st RT – first retention trial 24 h after the initial trial; 2nd RT – second retention trial 48 h after initial trial; 3rd RT – third retention trial after 7 days, initial trial. *; **; *** Significance of difference vs. vehicle-treated rats at p < 0.05, 0.01, and 0.001, respectively.
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tanol. The mixture was allowed to stand for 10 minutes, centrifuged and the butanol layer separated. Color intensity of the chromogen in the butanol was measured spectrophotometrically at 560 nm. The concentration of SOD was expressed as units/mg protein. • Statistical analysis: The data were processed by analysis of variance followed by post-test and individual comparisons using the Student’s t-test (two-tailed).
j Results Shuttle-box experiments
Of the aqueous, methanolic, chloroform and petroleum ether extracts (200 mg/kg body wt. each), only administration of the aqueous extract showed a significant increase in the number of avoidance responses in the initial trial, as the well as the first, second and third retention (Fig. 1). Therefore, subsequently only the aqueous extract was studied, using 3 different doses (100, 200 and 300 mg/kg body wt.). All three doses of the aqueous extract showed a significant increase in the number of avoidance response, to US in both initial and retention trails, but there was no significant difference among the three different doses tested (Fig. 2). Step-through experiments
The mean initial latency (IL) did not differ significantly between the control rats and the drug treatment groups. All the extracts at doses of 200 mg/kg
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body wt. prolonged the step-through latency in the first retention testing, as compared to controls. However, only the aqueous extract showed a statistically significant increase in step-through latency in both the second and third retention tests (Fig. 3). The
aqueous extract at the 3 different doses (100, 200 and 300 mg/kg body wt.) showed significant prolongation of retention latency, but the increase in the stepthrough latency did not differ significantly among the different doses tested (Fig. 4).
Fig. 2. Effect of aqueous extracts of Celastrus paniculatus (100, 200 and 300 mg/kg body wt.) on learning and memory in the shuttle-box. On the ordinate: mean number of avoidances (A) per 50 trials ± SEM of eight animals each; on the abscissa: IT- initial trial on day 7 of drug administration, 1st RT – first retention trial 24 h after initial trial; 2nd RT – second retention trial 48 h after the initial trial; 3rd RT – third retention trial after 7 days, initial trial. *; **; *** Significance of difference vs. vehicle-treated rats at p < 0.05, 0.01, and 0.001, respectively.
Fig. 3. Effect of aqueous, methanolic chloroform and petroleum ether extracts of Celastrus paniculatus at 200 mg/kg body wt. on learning and memory in the step-through apparatus. On the ordinate: mean stepthrough latency in sec ± SEM of ten animals each; on the abscissa: IL-initial trial on day 7 of drug administration, 1st RT – first retention latency 3 h after initial latency; 2nd RT – second retention latency 24 h after initial latency; 3rd RT – third retention latency after 7 days of initial latency. *; **; *** Significance of difference vs. vehicle-treated rats at p < 0.05, 0.01, and 0.001, respectively.
Antioxidant property of Celastrus paniculatus Willd. Step-down experiments
In the administration of the aqueous CPextract (100, 200 and 300 mg/kg body wt.), only the 200- and 300-mg/kg-treated rats showed significant increase in initial latency and in the retention tests at 3 h, 24 h and 7 days, as compared to the vehicle-treated group. There
Fig. 4. Effect of aqueous extracts of Celastrus paniculatus (100, 200 and 300 mg/kg body wt.) on learning and memory in the stepthrough apparatus. On the ordinate: mean step-through latency in sec ± SEM of ten animals each; on the abscissa: IL-initial trial on day 7 of drug administration, 1st RT – first retention latency 3 h after initial latency; 2nd RT – second retention latency 24 h after initial latency; 3rd RT – third retention latency after 7 days of initial latency. *; **; *** Significance of difference vs. vehicle-treated rats at p < 0.05, 0.01, and 0.001, respectively.
Fig. 5. Effect of aqueous extracts of Celastrus paniculatus asiatica (100, 200 and 300 mg/kg body wt.) on learning and memory in the stepdown apparatus. On the ordinate: mean step-down latency in sec ± SEM of ten animals each; on the abscissa: IL-initial trial on day 7 of drug administration, 1st RT – first retention latency 3 h after initial latency; 2nd RT – second retention latency 24 h after initial latency; 3rd RT – third retention latency after 7 days of initial latency. *; **; *** Significance of difference vs. vehicle-treated rats at p < 0.05, 0.01, and 0.001, respectively.
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was no significant difference between the two doses (200 and 300 mg/kg body wt.) (Fig. 5). Plus-maze experiments
The mean initial latency did not differ significantly between the vehicle- and drug-treated groups. Among
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Fig. 6. Effects of aqueous extracts of Celastrus paniculatus (100, 200 and 300 mg/kg body wt.) on learning and memory in the elevated-plus maze. On the ordinate: mean transfer latency in sec ± SEM of ten animals each; on the abscissa: ITL-initial transfer latency on day 7 of drug administration, 1st RT – first retention latency 24 h after initial latency; 2nd RT – second retention latency 7 days after initial latency. *; **; *** Significance of difference vs. vehicle-treated rats at p < 0.05, 0.01, and 0.001, respectively.
Table. 1 Effect of aqueous extracts of Celastrus paniculatus (100, 200 and 300 mg/kg body wt.) on MDA, glutathione, SOD and catalase levels in brains of normal rats on day 14. Each value represents mean ± SEM of ten animals each. *; **; *** Significance of difference vs. vehicle-treated rats at p < 0.05, 0.01, and 0.001, respectively. Treatment
Malondialdehyde (nmol/g tissue)
Control (Vehicle)
236.5 ± 20.2
Aqueous extract 100 mg/kg body wt. 200 mg/kg body wt. 300 mg/kg body wt.
219.9 ± 16.1 149.6 ± 13.1** 111.3 ± 7.9***
Glutathione (µg/g tissue) 88.6 ± 7.7 94.4 ± 10.5 138.8 ± 7.5*** 133.6 ± 14.5**
aqueous extracts when at 3 different doses only 200and 300-mg/kg body wt. showed an increase in the retention transfer latency, which is not a significant difference as compared to the vehicle-treated group in all the retention latency tests (Fig. 6). Estimation of markers of oxidative stress in the rat brain
When MDA, glutathione, SOD and catalase in rat brain were estimated on the day 14 of treatmeat with aqueous CP extract 100, 200 and 300 mg/kg body wt., the 100mg/kg dose caused no significant change in any parameters as compared to vehicle-treated rats. There was a sig-
Superoxide dismutase (Units/mg protein)
Catalase (Units/mg protein)
3.5 ± 0.3
16.68 ± 3.2
3.0 ± 0.5 4.2 ± 0.3 4.1 ± 0.5
24.08 ± 3.8 91.05 ± 14.7*** 92.50 ± 15.0***
nificant decrease in MDA level, and an increase in catalase and glutathione levels in the CP-treated group as compared to the vehicle-treated group, but there was no significant difference at the 200- and 300-mg/kg dose. There was an increase in SOD level in the 200- and 300mg/kg treated groups as compared to the vehicle-treated group, but the increase was not significant (Table 1).
j Discussion Aging and age-associated neurodegenerative diseases are associated with various degrees of behavioral impairments. The important candidates responsible for
Antioxidant property of Celastrus paniculatus Willd. producing the neuronal changes mediating these behavioral deficits are free radicals, which are responsible for the generation of oxidative stress (Maxwell, 1995; Cantuti et al., 2000). Thus, there is an increasing interest in the biochemical function of natural antioxidants contained in vegetables, fruits and medicinal plants, which could be candidates for prevention of oxidative damage and improvement of memory (Noda et al., 1997). Our attention has been focused, in particular on Celastrus paniculatus (CP). The different studies involving CPoil showed improvement in the different cognitive paradigms (Karanth et al., 1980; Karanth et al., 1981; Nalini et al., 1995; Gattu et al., 1997). Therefore in the present study different extracts of CP seeds were evaluated for their effects on learning and memory in normal rats, using four different paradigms. In the preliminary screening study, among the different extracts tested, only the aqueous extract at a dose of 200 mg/kg body wt. showed improvement in learning and memory tasks in both the shuttle-box and stepthrough paradigms. Therefore, the aqueous extract was evaluated in more detail using 3 doses (100, 200 and 300 mg/kg body wt.) in different learning and memory paradigms, i.e., shuttle-box, step-through, step-down and elevated-plus-maze. The three different doses of aqueous CP extract increased the number of avoidances in the shuttle-box and prolonged the step-through latency in the stepthrough apparatus, thus indicating facilitated learning and improved memory. There was no significant difference among the different doses tested. In the step-down paradigm only, animals treated with 200 and 300 mg/kg body wt. of the aqueous extract showed significant increase in step-down latency (the rat remained on the platform for a longer time), as compared to the vehicletreated group, indicating the acquisition of the stepdown task. In the elevated plus maze paradigm only the 200 and 300 mg/kg body wt. doses decreased the transfer latency as compared to the vehicle-treated group, but did not differ significantly. Thus, in the present study, the aqueous extract of CP seeds at doses of 100, 200 and 300 mg/kg body wt. was found to improve learning and memory in rats and the effect was more pronounced at 200 and 300 mg/kg without any dose-response relationship. This could be because the range of the doses used in this experiment might have been too narrow. The results of the present study provide scientific evidence to support reports in the Ayurvedic literature and confirm the ability of chronic administration of CP to enhance the performance of subjects engaged in memory-related tasks. The mechanism by which CP enhances learning and memory performance in behavioral tasks is yet unknown. Reports suggest that the varying degrees of behavioral impairments are associated with aging and age-
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associated neurodegenerative diseases (Reiter, 1995) and oxidative stress due to free radicals is responsible for producing the neuronal changes mediating these behavioral deficits (Cantuti et al., 2000). This refers to the cytotoxic consequences of oxygen radicals like superoxide anion, hydroxyl radical, and hydrogen peroxide, which act on polyunsaturated fatty acids in brain, thereby propagating the lipid peroxidation (Coyle and Puttfarcven, 1993). The major antioxidant and oxidative free-radical scavenging enzymes are glutathione, SOD and catalase. Antioxidant therapy includes either natural antioxidant enzymes or agents, which are capable of augmenting the functions of these enzymes (Bast et al., 1991). Earlier reports have shown that natural drugs like Ginkgo biloba (Christen, 2000; Sastre et al., 2000) and Withania somnifera (Bhattacharya et al., 1996), which improve cognition, also have antioxidant properties. Therefore, the different doses of aqueous extract (100, 200 and 300 mg/kg body wt.) of CP, which showed improvement in learning and memory paradigms, were tested on the oxidative stress parameters, i.e., levels of MDA, glutathione, SOD and catalase in brains of adult rats. Among the different doses of aqueous extract tested, only 200- and 300-mg/kg body wt. treated rats showed a significant decrease in the brain levels of malondialdehyde, which is the measure of lipid peroxidation and free radical generation. There was a simultaneous, significant increase in levels of glutathione, a tripeptide found in all cells which reacts with free radicals to protect cells from superoxide radical, hydroxyl radical and singlet oxygen (Schulz et al., 2000). In the present study there was a significant increase in levels of catalase at doses of 200 and 300 mg/kg body wt. as compared to the vehicletreated group, indicating that the aqueous extract scavenges the hydrogen peroxide, and there was an insignificant increase in levels of SOD, which is the only enzyme that uses the superoxide anions as a substrate and produces hydrogen peroxide as a metabolite, which is more toxic than O2 radical and has to be removed by catalase (Harman, 1991; Carrillo et al., 1992). These results suggest that the aqueous extract of CP reduces oxidative stress by increasing the endogenous antioxidant enzymes. The result of this study is in accordance with the reports of Russo et al. (2001), who showed that CP has antioxidant properties in in vitro studies. The present study therefore demonstrates that aqueous CP seed extract has two pronounced effects, i.e., improving learning and memory and antioxidant properties by decreasing the lipid peroxidation, and augmenting endogenous antioxidant enzymes in brain. The mechanism by which CP enhances cognition can be attributed at least in part to antioxidant properties. The serotonergic receptor antagonist property (Gattu et al., 1997) or ability to decrease the levels of norepinephrine, dopamine and serotonin,
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and its metabolites in the brain (Nalini et al., 1995), can be other mechanisms. Acknowledgments
We are thankful to Dabur Research foundation for their help in preparing extracts, and to the Council for Scientific and Industrial Research for a fellowship to M. H. Veerendra Kumar.
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Ellman, G. L.: Tissue sulfhydryl groups, Arch. Biochem. Biophy. 82: 70–77, 1959. Gassen, M. and Youdin, M. B.: The potential role of iron chelators in the treatment of Parkinson’s disease and related neurological disorders. Pharmacol. Toxicol. 80: 159–166, 1997. Gattu, M., Boss, K. L., Alvin, V., Terry, J. R., and Buccafusco, J.: Reversal of scopolamine induced deficits in navigational memory performance by the seed oil of Celastrus paniculatus. Pharmacol. Biochem. Behav. 57(4): 793–799, 1997. Good, P. F., Werner, P., Hsu, A., Olanow, C. W. and Perl, D. P.: Evidence of neuronal oxidative damage in Alzheimer’s disease. Am. J. Pathol. 149: 21–28; 1996. Harman, D.: The aging process: Major risk factor for disease and death. Proc. Natl. Acad. Sci. 88: 5360–5371, 1991. Jainkang, L., Rei, E., Hideaki, K. and Akitane, M.: Antioxidant action of guilingi in the brain of rats with FeCl3 induced epilepsy. Free Radi. Bio. Med. 9: 451–454, 1990. Joglaker, G. and Balwani, T. H., Review of tranquilizing effects of Celastrus paniculatus, J. Ind. Med. Assoc. 1: 190–195, 1967. Kakrani, H. K., Vijayanathan, N. G., Kalyani, G. A., and Satyanarayana, D.: Studies on Ayurvedic drugs I. Evaluation of anti-fatigue effect of the Ayurvedic drug ”Alert” in rats. Fitoterapia 56: 293–295,1985. Kakkar, P., Das, B. and Viswanathan, P. N.: A modified spectrophotometric assay of superoxide dismutase. J. Biochem. Biophys. 21: 130–132, 1984. Karanth, K. S., Haridas, K. K., Gunasundari, S., and Guruswami, M. N.: Effect of Celastrus paniculatus on learning process. Arogya 6: 137–139, 1980. Karanth, K. S., Padma, T. K., and Gunasundari, M. N.: Influence of Celastrus oil on learning and memory. Arogya 7: 83 –86, 1981. Khanna, A. K., Chander, R., and Kapoor, N. K.: Hypolipidemic activity of Abana in rats. Fitoterapia 62: 271–274, 1991. Lowry, O. H., Roseburgh, N. J., Farr, A. L. and Randal, R. J.: Protein measurement with the folin phenol reagent, J. Biol. Chem. 193: 205–215, 1951. Maxwell, S.R.: Prospects for the use of antioxidant therapies. Drugs 49: 345–361, 1995. Nalini, K., Aroor, A. R., Kumar, and K. B., Rao, A., Studies on biogenic amines and their metabolites in mentally retarded children on Celastrus oil therapy. Alternative Med. 1: 355–360, 1986. Nalini, K., Karanth, K. S., Rao, A., and Aroor, A. R.: Effects of Celastrus paniculatus on passive avoidance performance and biogenic amine turnover in albino rats. J. Ethnopharmacol. 17: 101–108, 1995. Noda, Y., Anzai, K., Mori A., Kohono, M., Shimnei, M., and Packer, L.: Hydroxyl and superoxide anion radical scavenging activities of natural source antioxidants using computerized JES-FR30 ESR spectrometer system. Biochem. Mol. Biol. Internat. 42: 35–44, 1997. Patel, P. P. and Trivedi, B. M.: The in vitro antibacterial activity of some medicinal oils. Ind. J. Med. Res. 50: 218 – 222, 1962. Reiter, R. J.: Oxidative processes and antioxidative defence mechanisms in the aging brain. FASEB 9: 526–533, 1995. Russo, A., Izzo, A., Cardile, V., Borelli F. and Vanella, A.: Indian medicinal plants as antiradicals and DNA cleavage protectors. Phytomedicine 8(2):125–132; 2001.
Antioxidant property of Celastrus paniculatus Willd. Sastre, J., Pastardo, F. V. and Vina, J.: Mitochondria, oxidative stress and aging. Free Rad. Res. 32(3): 189–198, 2000. Schulz, J. B., Linderau, J. and Dichgans, J.: Glutathione, oxidative stress and neurodegeneration. Eur. J. Biochem. 276(16): 4904–4911, 2000. Vaidyaratnam, P. S. V.: Indian Medicinal Plants: a compendium of 500 species, Vol. 2, Orient Longman Ltd., Anna Salai, Madras, India, p. 47–51, 1994. Vesselin, D. P., Ruman, K., Stiljana, B., Elena, K., Vesselin, V. P., Damjanka, G. and Vietaria, M.: Memory effects of standardized extracts of Panax ginseng (G115), Ginkgo biloba (GK501) and their combination Gincosan (PHL00701). Planta Med. 59: 106–114, 1993.
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j Address Prof. Y. K. Gupta, Neuropharmacology Laboratory, Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110029 Tel.: ++91-11-6593684; Fax: ++91-11-6864789; e-mail: [email protected]
Phytomedicine
Antioxidant property of Celastrus paniculatus Willd.: a possible mechanism in enhancing cognition M. H. V. Kumar and Y. K. Gupta Neuropharmacology laboratory, Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India
Summary In the present study aqueous, methanolic, chloroform and petroleum ether extracts of seeds of Celastrus paniculatus were investigated for their effect on cognitive functions in rats. Male Wistar rats weighing 200–250 g each were used to study effect on learning and memory through use of the shuttlebox, step-through, step-down and elevated plus maze paradigms. Only the aqueous seed extract (200 mg/kg body wt. for 14 days) showed an improvement in learning and memory in both the shuttle-box and step-through paradigms. Therefore, further experiments were conducted using the aqueous extract at 100, 200 and 300 mg/kg body wt. doses in different paradigms of cognition. All three doses of the aqueous extract increased the number of avoidances in the shuttle-box and step-through latency the in step-through apparatus, but no significant difference was observed between the doses tested. In the step-down apparatus, the 200- and 300-mg/kg body wt. doses of aqueous extract showed a significant increase in step-down latency, whereas no significant difference was observed in the elevatedplus-maze paradigm between drug-treated and vehicle-treated groups. Since the behavioral impairments are associated with oxidative stress, we investigated the effect of the aqueous extract on oxidative stress parameters. Among the three doses tested, only 200 and 300 mg/kg body wt. stimulated a significant decrease in the brain levels of malondialdehyde, with simultaneous significant increases in levels of glutathione and catalase. The present findings indicate that the aqueous extract of Celastrus paniculatus seed has cognitive-enhancing properties and an antioxidant effect might be involved. Key words: Celastrus paniculatus, learning and memory, active avoidance, passive avoidance, antioxidant
j Introduction Ayurveda, the ancient traditional system of medicine has described Celastrus paniculatus Willd. (CP) (Celastraceae) for prevention and treatment of a wide number of disease. According to Ayurveda, CP may be employed as a stimulant nervine tonic, rejuvenant, sedative, tranquilizer and diuretic. It is also used in the treatment of leprosy, leucoderma, rheumatism, gout, paralysis and asthma (Vaidyaratnam, 1994). Sporadic reports in the scientific literature also exist to suggest that the oil of CP and its extracts exhibit the following actions: antiviral (Bhakuni et al., 1969), antibacterial (Patel and Trivedi, 1962), insecticidal (Atal et al.,
1978), analgesic, anti-inflammatory (Ahmad et al., 1994; Dabral and Sharma, 1983), antimalarial (Ayudhaya et al., 1987), antifatigue (Kakrani et al., 1985), antispermatogenic (Wangoo and Bidwai, 1988), hypolipemic (Khanna et al., 1991), sedative (Ahumada et al., 1991) and anticonvulsant (Joglaker and Balwani, 1967). The indigenous people employ CP also as a general cognitive-enhancing agent. Several laboratory reports are now available to support these uses. Karanth et al. (1980), Karanth et al. (1981), and Nalini et al. (1995), reported that rats treated with CP oil improved their performance in the raised-platform shock avoidance 0944-7113/02/09/04-302 $ 15.00/0
Antioxidant property of Celastrus paniculatus Willd. and two-way passive avoidance paradigms. Nalini et al. (1986) also reported that chronic treatment with CP oil produced improvement in I. Q. scores and decreased the content of catecholamine metabolites in mentally retarded children. However, a comparative study of the different extracts of the seed on paradigms of learning and memory is lacking. Therefore, in the present study the aqueous, methanolic, chloroform and petroleum ether extracts of CP seed were evaluated in different learning and memory paradigms in rats. Reactive oxygen species (ROS) from both endogenous and exogenous sources are involved the aging and age-associated neurodegenerative disease, and reports suggest that ROS are associated with cognitive deficits (Good et al., 1996; Gassen and Youdim, 1997). Recently Russo et al. (2001) have reported that CPshows antioxidant effects in in vitro tests. Therefore, the effect of the CPextract which showed the maximum cognitiveenhancing property was also studied in markers of brain oxidative stress, namely, malondialdehyde (MDA), glutathione, superoxide dismutase (SOD) and catalase.
j Materials and Methods Animals
Studies were carried out using male Wistar rats weighing 200–250 g each. They were obtained from the central animal facility of the All India Institute of Medical Sciences, New Delhi, and stock bred in the departmental animal house. The rats were group-housed in polyacrylic cages (38 × 23 × 10 cm) with not more than 4 animals per cage and maintained under standard laboratory conditions (temperature 25 ± 2 °C) with a light and dark cycle (14 ± 1 h: 10 ± 1 h). They were allowed free access to standard dry pellet diet (Golden Feeds, India) and tap water ad libitum. All behavioral tests were performed between 9:00 and 13:00 h. All procedures described were reviewed and approved by the Institutional Committee for Ethical use of Animals. Plant material
Seeds of CP were procured from the local commercial market of New Delhi. The samples were then authenticated for their correct botanical identity by the chief Botanist, Department of Drug Standardization, Dabur Research Foundation, Ghaziabad, India. The whole plant was dried and coarsely ground. For the preparation of the aqueous extract, coarse powder of the seed was extracted with 8 parts boiling water for 5 h and filtered to yield the extract. The extract was then concentrated and finally dried to a powder. For the methanolic, chloroform and petroleum ether extracts, the coarse powder of the seed was extracted with 5 parts methanol,
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chloroform or petroleum ether at 60–65 °C for 5 h and filtered to yield the extracts. The extracts were then concentrated in vacuo and concentration was continued to completely remove the solvent. The percentage (w/w) yields of the aqueous, methanolic chloroform and petroleum ether extracts were 8, 24.8, 25.6 and 20 percent, respectively. Treatment schedules
The animals were divided in two sets of 5 groups to test the 4 extracts (aqueous, methanolic, chloroform and petroleum ether) and the vehicle on the shuttle-box and step-through apparatus paradigms . In both the sets, the 5 groups of animals were administered vehicle orally (Tween 80:water (1:5)), or aqueous extract, methanolic extract, chloroform extract or petroleum ether extract, each at 200 mg/kg body wt.for 14 days. One set was tested on the shuttle-box and the number of avoidances to unconditioned stimulus (US) was noted. Another set was tested on the step-through apparatus and the prolongation of step-through latency was determined. Of the 4 extracts tested, the aqueous extract showed a significant improvement in learning and memory in both the shuttle-box and step-through paradigms. Therefore, further experiments were conducted using the three doses (100, 200 and 300 mg/kg body wt.) of only the aqueous extract in the shuttle-box, step-through apparatus, step-down apparatus and elevated-plus-maze paradigms. The rats were divided into 3 sets of 4 groups each. The 4 groups of all three sets were administered vehicle (Tween 80:Water (1:5)), or aqueous extract at 100, 200 and 300 mg/kg body wt., respectively, for 14 days. The first set of animals was tested on the shuttle-box and the number of avoidances to US was noted. The second set was tested on the step-through paradigm and prolongation of step-through latenc was noted. In the third set of experiments, two memory paradigms were tested. First, in the step-down apparatus to note the step-down latency, followed by the elevated-plus-maze paradigm, to determine the effect on transfer latency. Following the, behavioral test on the fourteeth day, the animals of the third set were sacrificed for estimation of markers of oxidative stress. Behavioral test
• Two-way active avoidance with negative (punishment) reinforcement: Shuttle-box: The apparatus was a box (29 × 23 × 21 cm) divided into two equal compartments with an opening of 8 × 8 cm in middle of the wall separating the 2 compartments. A light source (25 W bulb) and a buzzer (70 db) were fitted in both compartments and switched on alternatively in the two compartments. These were used as conditioned stimulus (CS). The unconditioned stimulus (US) was an electric shock
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(50 V AC) applied to the grid floor for 2 s. Each trial consisted of conditioned stimulus for 10 s, followed by unconditioned stimulus of 2 s electric shock. The intertrial interval was 8 s. Thus, the total time for one trial was 20 s. An avoidance response was recorded when the animal avoided the US (i.e., animals crossed to the other compartment) within 10 s after the onset of CS. The rats were treated orally through an intragastric feeding tube with the drugs for 14 days. On the sixth day each rat was adapted to the shuttle-box. For this purpose, each rat was placed in a compartment of the apparatus and 20 conditioned stimuli (12-s light & buzzer and 8-s interval) were applied without electrical reinforcement. On the seventh day, one h after the administration of the drugs, each rat was trained with 50 such trials called “initial trials”. The avoided response (A) and unavoided response (UA) were recorded. Retention tests were given 24 h, 48 h and 7 days after the initial trial and termed as the first, second and third retention trials (RT), respectively (Vesselin et al., 1993). • Passive avoidance with negative (punishment) reinforcement: Step-through: The apparatus consisted of equal-size light and dark compartments (30 × 20 × 30 cm). A 40-watt bulb was fixed 30 cm above its floor, in the center of the roof of the light compartment. The floor consisted of a metal grid connected to a shock scrambler. The two compartments were separated by a trap door that could be raised to 10 cm. The rats were treated orally through an intragastric feeding tube for 14 days. On the seventh day of drug administration, rats were placed in the light compartment and the time taken to enter the dark compartment, with all four paws inside it, was recorded and termed as initial latency (IL). Immediately after the rat entered the dark chamber, with all four paws inside the dark chamber, the trap door was closed and an electric foot shock (50 V AC) was delivered for 2 s. Rats that had an initial latency of more than 60 s were excluded from experiments. 3 h, 24 h and 7 days later, the latency (time lapse before each animal entered the dark compartment) was recorded in the same manner as in the acquisition trial and were termed as first, second and third retention latencies (RL), respectively, but the foot shock was not delivered, and the latency time was recorded to a maximum of 600 s (Vesselin et al., 1993). • Passive avoidance with negative (punishment) reinforcement: step-down: The apparatus consisted of a chamber (29 × 30 × 29 cm) with a metal grid floor. A wooden platform (10 × 8 cm) was fixed in the center of the grid floor. The rats were treated orally through a probe for 14 days. On the seventh day of drug treatment, the rats were placed on the platform and trained to remain on it for a longer time to escape the electric footshock. Each descent of the animal to the grid floor was punished by electric footshock for 2 s (50 V a.c).
Each training session consisted of 5 trials. Time taken to descent in the last trial (fifth) was considered as “Initial latency” (IL). Retention latencies were tested 3 h, 24 h and 7 days later by placing the animal onto the platform again and measuring the time of descent and were termed as first, second, and third retention latencies (RL), respectively (Vesselin et al., 1993). • Elevated Plus Maze: The plus maze consisted of two opposite open arms (50 × 10 cm), crossed with two closed arms of the same dimensions, with 40-cm high walls. The arms were connected with a central square (10 × 10 cm). On day 7 of drug treatment, rats were placed individually at one end of an open arm, facing away from the central square. The time taken for the rat to move from the open arm and enter into one of the closed arms was recorded and termed as “transfer latency” (TL). Each animal was allowed to explore the maze for 30 sec after recording transfer latency. After 24 h and 7 days, the rat was placed similarly on the open arm and retention latency (RTL) was noted again (Bhattacharya, 1994). While doing the experiments with the shuttle-box, step-through and step-down apparatus, the grid as well and the rat paw were moistened with water before delivering foot shock, as it is known to reduce the wide interanimal variability in paw-skin resistance among rats. Biochemical estimation of markers of oxidative stress
On day 14 following the behavioral testing, animals were decapitated under ether anesthesia and the brains quickly removed, cleaned with ice-cold saline and stored at –80 °C. • Tissue preparation: Brain-tissue samples were thawed and homogenized with 10 times (w/v) homogenizer in ice-cold 0.1 M phosphate buffer (pH 7.4). Aliquots of homogenates from the rat brains were separated and used to measure protein, lipid peroxidation and glutathione. The remaining homogenates were centrifuged at 15,000 rpm for 60 min and the supernatant was then used for enzyme assays. Catalase activity was determined immediately after sample preparation and superoxide dismutase was determined within 24 h. Protein concentration was determined according to Lowry et al. (1951) using purified bovine serum albumin as a standard. • Estimation of Malondialdehyde: Malondialdehyde (MDA), a measure of lipid peroxidation, was estimated as described by Jainkang et al. (1990). The reagents acetic acid 1.5 ml (20 %), pH 3.5, 1.5 ml thiobarbituric acid (0.8 %) and 0.2 ml sodium dodecyl sulphate (8.1 %) were added to 0.1 ml of processed tissue samples, then heated at 100 °C for 60 min. The mixture was cooled under tap water and 5 ml of n-butanol:pyridine (15:1), and 1 ml of distilled water was added. The mixture was vortexed vigorously. After centrifugation at 4000 rpm for 10 min,
Antioxidant property of Celastrus paniculatus Willd. the organic layer was separated and absorbance was measured at 532 nm using a spectrophotometer. The concentration of MDAis expressed as nmol/g tissue. • Estimation of glutathione: Glutathione was measured according to the method of Ellman (1959). An equal quantity of homogenate was mixed with 10 % trichloroacetic acid and centrifuged to separate the proteins. To 0.01 ml of this supernatant, 2 ml of phosphate buffer (pH 8.4), 0.5 ml of 5’5-dithiobis (2-nitrobenzoic acid), and 0.4 ml of double distilled water were added. The mixture was vortexed and the absorbance read at 412 nm within 15 min. The concentration of glutathione was expressed as µg/g tissue. • Estimation of catalase: Catalase activity was measured by the method of Aebi (1974). 0.1 ml of supernatant was added to a cuvette containing 1.9 ml of 50-mM phosphate buffer (pH 7.0). The reaction was started by the addition of 1.0 ml of freshly prepared 30-mM H2O2. The rate of decomposition of H2O2 was measured spectrophotometrically from changes in absorbance at 240 nm. The activity of catalase was expressed as units/mg protein. • Estimation of superoxide dismutase (SOD): The SOD activity of the brain tissue was analyzed by the method described by Kakkar et al. (1984). The assay mixture contained 0.1 ml of sample, 1.2 ml of sodium pyrophosphate buffer (pH 8.3, 0.052 M), 0.1 ml phenazine methosulphate (186 µM), 0.3 ml of 300 µM nitroblue tetrazolium, and 0.2 ml NADH (750 µM). Reaction was started by addition of NADH. After incubation at 30 °C for 90 sec, the reaction was stopped by the addition of 0.1 ml glacial acetic acid. The reaction mixture was stirred vigorously with 4.0 ml of n-bu-
Fig. 1. Effect of aqueous, methanolic, chloroform and petroleum ether extracts of Celastrus paniculatus (200 mg/kg body wt.) on learning and memory in the shuttle-box. On the ordinate; mean number of avoidances (A) per 50 trials ± SEM of eight animals each; on the abscissa: IT- initial trial on day 7 of drug administration, 1st RT – first retention trial 24 h after the initial trial; 2nd RT – second retention trial 48 h after initial trial; 3rd RT – third retention trial after 7 days, initial trial. *; **; *** Significance of difference vs. vehicle-treated rats at p < 0.05, 0.01, and 0.001, respectively.
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tanol. The mixture was allowed to stand for 10 minutes, centrifuged and the butanol layer separated. Color intensity of the chromogen in the butanol was measured spectrophotometrically at 560 nm. The concentration of SOD was expressed as units/mg protein. • Statistical analysis: The data were processed by analysis of variance followed by post-test and individual comparisons using the Student’s t-test (two-tailed).
j Results Shuttle-box experiments
Of the aqueous, methanolic, chloroform and petroleum ether extracts (200 mg/kg body wt. each), only administration of the aqueous extract showed a significant increase in the number of avoidance responses in the initial trial, as the well as the first, second and third retention (Fig. 1). Therefore, subsequently only the aqueous extract was studied, using 3 different doses (100, 200 and 300 mg/kg body wt.). All three doses of the aqueous extract showed a significant increase in the number of avoidance response, to US in both initial and retention trails, but there was no significant difference among the three different doses tested (Fig. 2). Step-through experiments
The mean initial latency (IL) did not differ significantly between the control rats and the drug treatment groups. All the extracts at doses of 200 mg/kg
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body wt. prolonged the step-through latency in the first retention testing, as compared to controls. However, only the aqueous extract showed a statistically significant increase in step-through latency in both the second and third retention tests (Fig. 3). The
aqueous extract at the 3 different doses (100, 200 and 300 mg/kg body wt.) showed significant prolongation of retention latency, but the increase in the stepthrough latency did not differ significantly among the different doses tested (Fig. 4).
Fig. 2. Effect of aqueous extracts of Celastrus paniculatus (100, 200 and 300 mg/kg body wt.) on learning and memory in the shuttle-box. On the ordinate: mean number of avoidances (A) per 50 trials ± SEM of eight animals each; on the abscissa: IT- initial trial on day 7 of drug administration, 1st RT – first retention trial 24 h after initial trial; 2nd RT – second retention trial 48 h after the initial trial; 3rd RT – third retention trial after 7 days, initial trial. *; **; *** Significance of difference vs. vehicle-treated rats at p < 0.05, 0.01, and 0.001, respectively.
Fig. 3. Effect of aqueous, methanolic chloroform and petroleum ether extracts of Celastrus paniculatus at 200 mg/kg body wt. on learning and memory in the step-through apparatus. On the ordinate: mean stepthrough latency in sec ± SEM of ten animals each; on the abscissa: IL-initial trial on day 7 of drug administration, 1st RT – first retention latency 3 h after initial latency; 2nd RT – second retention latency 24 h after initial latency; 3rd RT – third retention latency after 7 days of initial latency. *; **; *** Significance of difference vs. vehicle-treated rats at p < 0.05, 0.01, and 0.001, respectively.
Antioxidant property of Celastrus paniculatus Willd. Step-down experiments
In the administration of the aqueous CPextract (100, 200 and 300 mg/kg body wt.), only the 200- and 300-mg/kg-treated rats showed significant increase in initial latency and in the retention tests at 3 h, 24 h and 7 days, as compared to the vehicle-treated group. There
Fig. 4. Effect of aqueous extracts of Celastrus paniculatus (100, 200 and 300 mg/kg body wt.) on learning and memory in the stepthrough apparatus. On the ordinate: mean step-through latency in sec ± SEM of ten animals each; on the abscissa: IL-initial trial on day 7 of drug administration, 1st RT – first retention latency 3 h after initial latency; 2nd RT – second retention latency 24 h after initial latency; 3rd RT – third retention latency after 7 days of initial latency. *; **; *** Significance of difference vs. vehicle-treated rats at p < 0.05, 0.01, and 0.001, respectively.
Fig. 5. Effect of aqueous extracts of Celastrus paniculatus asiatica (100, 200 and 300 mg/kg body wt.) on learning and memory in the stepdown apparatus. On the ordinate: mean step-down latency in sec ± SEM of ten animals each; on the abscissa: IL-initial trial on day 7 of drug administration, 1st RT – first retention latency 3 h after initial latency; 2nd RT – second retention latency 24 h after initial latency; 3rd RT – third retention latency after 7 days of initial latency. *; **; *** Significance of difference vs. vehicle-treated rats at p < 0.05, 0.01, and 0.001, respectively.
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was no significant difference between the two doses (200 and 300 mg/kg body wt.) (Fig. 5). Plus-maze experiments
The mean initial latency did not differ significantly between the vehicle- and drug-treated groups. Among
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Fig. 6. Effects of aqueous extracts of Celastrus paniculatus (100, 200 and 300 mg/kg body wt.) on learning and memory in the elevated-plus maze. On the ordinate: mean transfer latency in sec ± SEM of ten animals each; on the abscissa: ITL-initial transfer latency on day 7 of drug administration, 1st RT – first retention latency 24 h after initial latency; 2nd RT – second retention latency 7 days after initial latency. *; **; *** Significance of difference vs. vehicle-treated rats at p < 0.05, 0.01, and 0.001, respectively.
Table. 1 Effect of aqueous extracts of Celastrus paniculatus (100, 200 and 300 mg/kg body wt.) on MDA, glutathione, SOD and catalase levels in brains of normal rats on day 14. Each value represents mean ± SEM of ten animals each. *; **; *** Significance of difference vs. vehicle-treated rats at p < 0.05, 0.01, and 0.001, respectively. Treatment
Malondialdehyde (nmol/g tissue)
Control (Vehicle)
236.5 ± 20.2
Aqueous extract 100 mg/kg body wt. 200 mg/kg body wt. 300 mg/kg body wt.
219.9 ± 16.1 149.6 ± 13.1** 111.3 ± 7.9***
Glutathione (µg/g tissue) 88.6 ± 7.7 94.4 ± 10.5 138.8 ± 7.5*** 133.6 ± 14.5**
aqueous extracts when at 3 different doses only 200and 300-mg/kg body wt. showed an increase in the retention transfer latency, which is not a significant difference as compared to the vehicle-treated group in all the retention latency tests (Fig. 6). Estimation of markers of oxidative stress in the rat brain
When MDA, glutathione, SOD and catalase in rat brain were estimated on the day 14 of treatmeat with aqueous CP extract 100, 200 and 300 mg/kg body wt., the 100mg/kg dose caused no significant change in any parameters as compared to vehicle-treated rats. There was a sig-
Superoxide dismutase (Units/mg protein)
Catalase (Units/mg protein)
3.5 ± 0.3
16.68 ± 3.2
3.0 ± 0.5 4.2 ± 0.3 4.1 ± 0.5
24.08 ± 3.8 91.05 ± 14.7*** 92.50 ± 15.0***
nificant decrease in MDA level, and an increase in catalase and glutathione levels in the CP-treated group as compared to the vehicle-treated group, but there was no significant difference at the 200- and 300-mg/kg dose. There was an increase in SOD level in the 200- and 300mg/kg treated groups as compared to the vehicle-treated group, but the increase was not significant (Table 1).
j Discussion Aging and age-associated neurodegenerative diseases are associated with various degrees of behavioral impairments. The important candidates responsible for
Antioxidant property of Celastrus paniculatus Willd. producing the neuronal changes mediating these behavioral deficits are free radicals, which are responsible for the generation of oxidative stress (Maxwell, 1995; Cantuti et al., 2000). Thus, there is an increasing interest in the biochemical function of natural antioxidants contained in vegetables, fruits and medicinal plants, which could be candidates for prevention of oxidative damage and improvement of memory (Noda et al., 1997). Our attention has been focused, in particular on Celastrus paniculatus (CP). The different studies involving CPoil showed improvement in the different cognitive paradigms (Karanth et al., 1980; Karanth et al., 1981; Nalini et al., 1995; Gattu et al., 1997). Therefore in the present study different extracts of CP seeds were evaluated for their effects on learning and memory in normal rats, using four different paradigms. In the preliminary screening study, among the different extracts tested, only the aqueous extract at a dose of 200 mg/kg body wt. showed improvement in learning and memory tasks in both the shuttle-box and stepthrough paradigms. Therefore, the aqueous extract was evaluated in more detail using 3 doses (100, 200 and 300 mg/kg body wt.) in different learning and memory paradigms, i.e., shuttle-box, step-through, step-down and elevated-plus-maze. The three different doses of aqueous CP extract increased the number of avoidances in the shuttle-box and prolonged the step-through latency in the stepthrough apparatus, thus indicating facilitated learning and improved memory. There was no significant difference among the different doses tested. In the step-down paradigm only, animals treated with 200 and 300 mg/kg body wt. of the aqueous extract showed significant increase in step-down latency (the rat remained on the platform for a longer time), as compared to the vehicletreated group, indicating the acquisition of the stepdown task. In the elevated plus maze paradigm only the 200 and 300 mg/kg body wt. doses decreased the transfer latency as compared to the vehicle-treated group, but did not differ significantly. Thus, in the present study, the aqueous extract of CP seeds at doses of 100, 200 and 300 mg/kg body wt. was found to improve learning and memory in rats and the effect was more pronounced at 200 and 300 mg/kg without any dose-response relationship. This could be because the range of the doses used in this experiment might have been too narrow. The results of the present study provide scientific evidence to support reports in the Ayurvedic literature and confirm the ability of chronic administration of CP to enhance the performance of subjects engaged in memory-related tasks. The mechanism by which CP enhances learning and memory performance in behavioral tasks is yet unknown. Reports suggest that the varying degrees of behavioral impairments are associated with aging and age-
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associated neurodegenerative diseases (Reiter, 1995) and oxidative stress due to free radicals is responsible for producing the neuronal changes mediating these behavioral deficits (Cantuti et al., 2000). This refers to the cytotoxic consequences of oxygen radicals like superoxide anion, hydroxyl radical, and hydrogen peroxide, which act on polyunsaturated fatty acids in brain, thereby propagating the lipid peroxidation (Coyle and Puttfarcven, 1993). The major antioxidant and oxidative free-radical scavenging enzymes are glutathione, SOD and catalase. Antioxidant therapy includes either natural antioxidant enzymes or agents, which are capable of augmenting the functions of these enzymes (Bast et al., 1991). Earlier reports have shown that natural drugs like Ginkgo biloba (Christen, 2000; Sastre et al., 2000) and Withania somnifera (Bhattacharya et al., 1996), which improve cognition, also have antioxidant properties. Therefore, the different doses of aqueous extract (100, 200 and 300 mg/kg body wt.) of CP, which showed improvement in learning and memory paradigms, were tested on the oxidative stress parameters, i.e., levels of MDA, glutathione, SOD and catalase in brains of adult rats. Among the different doses of aqueous extract tested, only 200- and 300-mg/kg body wt. treated rats showed a significant decrease in the brain levels of malondialdehyde, which is the measure of lipid peroxidation and free radical generation. There was a simultaneous, significant increase in levels of glutathione, a tripeptide found in all cells which reacts with free radicals to protect cells from superoxide radical, hydroxyl radical and singlet oxygen (Schulz et al., 2000). In the present study there was a significant increase in levels of catalase at doses of 200 and 300 mg/kg body wt. as compared to the vehicletreated group, indicating that the aqueous extract scavenges the hydrogen peroxide, and there was an insignificant increase in levels of SOD, which is the only enzyme that uses the superoxide anions as a substrate and produces hydrogen peroxide as a metabolite, which is more toxic than O2 radical and has to be removed by catalase (Harman, 1991; Carrillo et al., 1992). These results suggest that the aqueous extract of CP reduces oxidative stress by increasing the endogenous antioxidant enzymes. The result of this study is in accordance with the reports of Russo et al. (2001), who showed that CP has antioxidant properties in in vitro studies. The present study therefore demonstrates that aqueous CP seed extract has two pronounced effects, i.e., improving learning and memory and antioxidant properties by decreasing the lipid peroxidation, and augmenting endogenous antioxidant enzymes in brain. The mechanism by which CP enhances cognition can be attributed at least in part to antioxidant properties. The serotonergic receptor antagonist property (Gattu et al., 1997) or ability to decrease the levels of norepinephrine, dopamine and serotonin,
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and its metabolites in the brain (Nalini et al., 1995), can be other mechanisms. Acknowledgments
We are thankful to Dabur Research foundation for their help in preparing extracts, and to the Council for Scientific and Industrial Research for a fellowship to M. H. Veerendra Kumar.
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j Address Prof. Y. K. Gupta, Neuropharmacology Laboratory, Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110029 Tel.: ++91-11-6593684; Fax: ++91-11-6864789; e-mail: [email protected]