MODIFICATION OF NOOTROPIC ACTIVITY OF ALBIZZIA LEBBECK SAPONINS BY SEROTONERGIC AGENTS. Suvarna P.Ingale*, Sanjay B, Kasture #

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1 MODIFICATION OF NOOTROPIC ACTIVITY OF ALBIZZIA LEBBECK SAPONINS BY SEROTONERGIC AGENTS Suvarna P.Ingale*, Sanjay B, Kasture # *Assistant Professor, Dept. of Pharmacology, Indira College of Pharmacy, New Pune - Mumbai highway, Tathwade, Pune # Professor, Dept. of Pharmacology M.G.V Mandal s College Of Pharmacy, Panchvati, Nasik Summary The Objective of present study is to investigate the effect of various serotonergic agents on memory enhancing activity of Albizzia lebbeck. n- butanol soluble fraction of Albizzia Lebbeck (ALBF) was extracted and evaluated for nootropic activity. Three tests viz elevated plus maze test, object recognition test and passive shock avoidance test were used to evaluate nootropic activity. Different serotonergic agents like ondansetron, fluoxetine, cyproheptadine, buspirone, cisapride and mianserin were administered to animals to study involvement of different serotonergic receptors in learning and memory. n-butanol soluble fraction of Albizzia lebbeck (ALBF) showed nootropic activity. Ondansetron, Fluoxetine and cisapride when administered along with ALBF enhanced learning and memory while cyproheptadine, buspirone and mianserin impaired learning and memory indicating involvement of 5-HT receptors in learning and memory consolidation. It is concluded that the 5-HT 3 and 5-HT 4 receptors play an important role in learning and memory probably by releasing acetylcholine. Keywords: Albizzia lebbeck, Nootropic activity, Serotonergic agents, Introduction In India, the use of different parts of several medicinal plants to cure specific ailments has been in vogue from ancient times. India is perhaps the largest producer of medicinal herbs and is rightly called botanical garden of the world. In modern medicine also, plants occupy a very significant place as raw material for some important drugs. Several plants have been known to possess nootropic activity viz. red ginseng (1), Lowsonia inermis (2), Albizzia lebbeck (3), Clitoria ternatea (4), Bacopa monnieri (brahmi)(5)and Shilajit (6). Saponins, bacoside a and b from Bacopa monnieri and ginsenoside Rb1 and Rbinfluene, the saponins from Panax ginseng are the active principles for enhancing cognitive function (7). 891

2 Three main saponins named albiziasaponin A(acacic acid lactone 3-O-β-D-xylopyranosyl-(1 2)-α-L-arabinopyranosyl-(1 6)- β -D-glucopyranoside), albiziasaponin B(3-O- β -Dglucopyranosyl-(1 2)-O-( α -L-arabinopyranosyl-(1 6))- β-d-glucopyranoside), and albiziasaponin C (-O-β-D-xylopyranosyl-(1 2)-α-L-arabinopyranosyl-(1 6)-O-( β -Dglucopyranosyl-(1 2) )- β -D-glucopyranoside) were isolated from the barks of Albizia lebbeck (8). Albizziahexoside, a new hexaglycosylated saponin was isolated from leaves of Albizia lebbeckn (9). Till date Albizzia lebbeck has been studied for anti-inflammatory (10), anticonvulsant (11), anti-diarrhoeal (12), anti-tumour(13), antimicrobial(14) immunomodulatory(15), anxiolytic and nootropic activities (16).Previous study from this laboratory has reported that saponins containing fractions of Albizzia lebbeck (ALBF) has nootropic activity. Therefore we have studied nootropic activity of Albizzia lebbeck using elevated plus maze test, passive shock avoidance test and object recognition test and further modification of its nootropic activity by different serotonergic agents. The different serotonergic agents such as ondansetron (5-HT 3 antagonist), cyproheptadine ((5- HT 2A antagonist), fluoxetine (selective serotonin reuptake inhibitor), buspirone (partial 5-HT 1a agonist), cisapride ((5-HT 4 agonist), and mianserin (5-HT 2 and 5-HT 1c antagonist) in a single doses modifying serotonergic function, based on literature were used. Materials and Methods Animals Swiss albino mice (Serum Institute of India, Pune) weighing between 20-25g of either sex were used. Animals were housed under standard conditions of temperature (24±2 C) and relative humidity (30-70%) with a 12:12 light: dark cycle. The animals were fed with standard pellet diet and water ad libitum. The Institutional Animal Ethics Committee (IAEC) of the N.D.M.V.P.Samaj s College of Pharmacy, Nasik-2 registered under CPCSEA India (Reg no. CPCSEA/ 141) approved the study. Nine groups each containing five animals was employed. Extraction The plant Albizzia lebbeck was collected fresh from campus of N.D.M.V.P.Samaj s College of Pharmacy, Nasik-2 and authenticated by Department of Pharmacognosy. Saponins were isolated using method described by Pal et al (8). Shade dried powered bark (730g) of Albizzia lebbeck was defatted with petroleum ether (2 litres, C) and was extracted with methanol using Soxhlet apparatus. The methanol extract was evaporated to dryness. This residue (27g) was suspended in water, extracted successively with ethyl acetate and n-butanol. n-butanol soluble fraction (ALBF) was tested for the presence of saponins and used for pharmacological study. ALBF was used in a single dose of 0.1 mg/kg depending on the observations of previous study done on Albizzia lebbeck by Brahmankar et al (3). Drugs: Name of drug Piracetam Ondansetron Fluoxetine Buspirone Cyproheptadine Cisapride Mianserin Source Uni-UCB Ltd, India Cipla Ltd, India M. J. Pharmaceuticals Ltd, India Cadila Labs, India Themis Chemicals Ltd, India Torrent Pharmaceuticals Ltd, India Torrent Pharmaceuticals Ltd, India 892

3 Experimental 1) Elevated plus maze test (17) An elevated plus maze consisting of two open arms (36x6cm) and two enclosed arms (35x6x15cm) was used. The maze was elevated at the height of 25cm. Mice were placed individually at the end of the open arm and the time taken by the animal to enter into either of the enclosed arm (transfer latency) was recorded. On the first day the mouse was allowed to explore the plus maze for 5 min and sent back to home cage after the first trial. After 24 hours mice were placed again on the elevated plus maze individually as before and transfer (TL) was noted again. TL measured on first day served as parameter for acquisition. TL was expressed as retention scores after 24 hours or one week for each mouse by calculating the inflexion ratio. Inflexion ratio= (L1-L0)/L0 Where L0=transfer latency after 24 hours/week in seconds. L1=initial transfer latency in seconds. The animal received vehicle orally sixty minutes before the first trial whereas ALBF piracetam, ondansetron, fluoxetine, cyproheptadine, buspirone, cisapride and mianserin were administered i.p. ALBF was administered 30min. before the other drugs and the trial was performed 30 min. later. No treatment was given on day 2 nd and 9 th day. Each group consisted of 5 animals. 2) Object recognition test (18) The apparatus was formed by the colored plywood (70x60x30cm) with a grid floor that could be easily cleaned with hydrogen peroxide after each trial. The apparatus was illuminated by 60 W lamp suspended 50cm above the box. The objects to be discriminated were also made of plywood in two different shapes of 8cm height colored black. The day before test mice were allowed to explore the box (without any object) for two min. On the day of test in the first trial (T 1 ) two identical objects were placed in two opposite corners of the box and the amount of time taken by each mouse to complete 20 sec of object exploration was recorded. Exploration was considered directing the nose at a distance <2cm to the object and /or touching it with the nose. During the second trial (T 2, 90 min after T 1 ) one of the objects presented in trial T 1 was replaced by new object and the mice were left in the box for 5 min. The time spent for exploration of familiar (F) and the new object (N) were recorded separately and discrimination index (D) was calculated (N-F/N+F). Care was taken to avoid place preferences and olfactory stimuli by randomly changing the role (familiar or new object) and the position of the two objects during T 2 and cleaning the apparatus with hydrogen peroxide. The animal received vehicle orally sixty minutes before the first trial whereas ALBF piracetam, ondansetron, fluoxetine, cyproheptadine, buspirone, cisapride and mianserin were administer i.p. ALBF was administered 30 min. before other drugs and the first trial (T 1 )was performed 30 min. later. The second trial was performed 90 min. after the first trial. 3) Passive shock avoidance test (2) The apparatus consisted of an electric grid (24x30cm.) with a shock free zone (2x3x1cm) in the center and the entire grid having perflex enclosure. The mice were placed individually on electric grid and allowed to explore for one min. The stimulus (20V) with AC current of 5mA was then applied and latency to reach SFZ was recorded three consecutive times as basal reading. Animals that reached the SFZ within 2 min. in first trial were selected for the study. After one hour of the first trial, each animal was placed on the grid again. The stimulus (20V) with AC current of 5mA was then applied and latency to reach SFZ and number of mistakes 893

4 animal made in 15 min. were recorded as parameter for acquisition and retention respectively. The animal received vehicle orally sixty minutes before the first trial whereas ALBF piracetam, ondansetron, fluoxetine, cyproheptadine, buspirone, cisapride and mianserin were administer i.p ALBF was administered 30 min. before other drugs.the second trial was performed 60 min. after the first trial. Statistical analysis The observations are mean ±SEM. Significant differences were evaluated using ANOVA followed by Dunnett s test and Student t test. Results In elevated plus maze test, the transfer latency TL measured on day 1, day 2 and day 9.TL was expressed as inflexion ratio (IR).As TL decreases IR increases. Piracetam (100mg/kg, i.p.)and ALBF (0.1mg/kg,) showed significant decrease in TL on day 2 and day 9 as compared to vehicle treated group (P< )., Ondansetron (0.1mg/kg, i.p.), Fluoxetine (5mg/kg, i.p.) and Cisapride (10mg/kg, i.p.) administered along with ALBF (0.1mg/kg,) showed significant decrease in TL on day 2 and day 9 as compared to ALBF treated group (P< ).Cyproheptadine (10mg/kg, i.p.)and Mianserin (10mg/kg, i.p.) administered along with ALBF (0.1mg/kg,) showed significant increase in TL on day 2 and day 9 as compared to ALBF treated group (P< ). Buspirone (5mg/kg, i.p.) administered along with ALBF (0.1mg/kg,) showed significant decrease in TL on day 2 (P< ) while TL was increased on day 9 as compared to ALBF treated group. In passive shock avoidance test, latency to reach SFZ and mistakes made by animal in 15 min were measured as parameter for acquisition and retention respectively. Piracetam (100mg/kg, i.p.) and ALBF (0.1mg/kg) showed decrease in latency to reach SFZ and significant decrease in mistakes made in 15 min as compared to vehicle treated group (P<). Ondansetron (0.1mg/kg),Buspirone (5mg/kg), Fluoxetine (5mg/kg) and Cisapride (10mg/kg) administered along with ALBF (0.1mg/kg) showed decrease in latency to reach SFZ and significant decrease in mistakes made in 15 min as compared to control group (P<). Cyproheptadine (10mg/kg) and Mianserin (10mg/kg) administered along with ALBF (0.1mg/kg) showed significant increase in latency to reach SFZ (P<) and decrease in mistakes made in 15 min. In object recognition test, the time spends for exploration of familiar and new object was measured and the discrimination index was calculated as parameter for acquisition and retention. Piracetam (100mg/kg, i.p.) and ALBF (0.1mg/kg, i.p.) showed increase in discrimination index D compared to vehicle treated group. Piracetam and ALBF both showed significant increase in time spent for exploration of new object compared to familiar object (P<). Ondansetron (0.1mg/kg, i.p.), Fluoxetine (5mg/kg, i.p.) and Cisapride (10mg/kg, i.p.)administered along with ALBF (0.1mg/kg, i.p.) increased discrimination index D compared to control group. The time spends for exploration of new object was increased significantly compared to familiar object (P<). Buspirone (5mg/kg, i.p.) administered along with ALBF decreased discrimination index D compared to control group. Cyproheptadine (10mg/kg, i.p.)and Mianserin (10mg/kg, i.p.), administered along with ALBF restored discrimination index D compared to control group. 894

5 Table 1: Effect of piracetam and n-butanolic fraction of Albizzia Lebbeck on transfer latency in elevated plus maze Treatment Transfer latency Inflexion ratio mg/kg Day1 Day2 Day9 Day2 Day9 Vehicle (5ml/kg) Piracetam (100mg/kg) ALBF (0.1mg/kg) Ond + ALBF ( ) Cyp + ALBF ( ) Mian + ALBF ( ) Busp + ALBF ( ) Fluox +ALBF ( ) Cisap +ALBF ( ) One way ANOVA F P 62.80± ±0.86 # 15.2±1.59 # 4.26± ± ± ±0.96 #* 5.8±0.58* # 5.55± ± ±0.47* 5.0±1.26 #* 5.8±0.37* # 7.58± ± ± ±0.4 # 2.8±0.37 # 9.9± ±1.48* 216.8±31.8* 57±0.70* # 52.6±1.50* # 2.8± ± ±17.48* 92.2±1.01* # 77.8±1.28* # 1.80±0.21* 2.31± ±5.83* 2.9±1.51 # 24.6±1.32* # 3.49± ± ± ±0.37 # 4.2±0.37 # 10.99± ±1.03* 30.2± ±0.4 # 4.2±0.58 # 11.93± ±1.24* Values are mean ± SEM, n=5 in each group *P< compared to respective control (ANOVA followed by Dunnett test) # P< compared to day 1(Student t test) 895

6 Table 2: Effect of Piracetam and n-butanolic fraction of Albizzia lebbeck on latency to reach SFZ in Passive shock avoidance test Treatment (mg/kg) Latency to reach SFZ (seconds) Mistakes in 15 minutes Vehicle (5ml/kg) 9± ±1.029 Piracetam (100mg/kg) 4.4± ±1.593* ALBF (0.1mg/kg) 4.8± ±2.14* Ond +ALBF ( ) 2.6 ± ± 1.095* Cyp + ALBF ( ) 71 ± 16.2* 10.6 ± 0.67 Mian + ALBF ( ) 28 ± 6.67* 6.75 ± 0.76 Busp+ALBF (5+ 0.1) 8.8± ± 1.5 Fluox+ ALBF (5+ 0.1) 3.6± ± 0.37* Cisap+ ALBF ( ) 4.2± ± One way F ANOVA P Values are mean ± SEM, n=5 in each group * P< compared to respective control (ANOVA followed by Dunnett test) 896

7 Table 3: Effect of Piracetam and n-butanolic fraction of Albizzia lebbeck on T 1 andt 2 session in object recognition test Treatment (mg/kg) T 1 session T 2 session (sec) D (sec) F N Vehicle (5mg/kg) 234.6± ± ± ±0.043 Piracetam(100mg/kg) 225.6± ±4.12* 29.2±2.87 # 0.136±0.044 ALBF (0.1mg/kg) 250.4± ±1.019* 22.2±3.58 # 0.44±0.04* Ond+ALBF ( ) 149.6±19.25* 13.2± ±3.92 # 0.46±0.096 Cyp+ALBF( ) 261.2± ± ±11.44 # * 0.44±0.05 Mian+ ALBF( ) 235± ± ±372 # * 0.44±044 Busp+ALBF(5+ 0.1) 264± ±9.52* 147.6±16.43* # 0.36±0.09 Fluox+ALBF(5+ 0.1) 148.7±9.77* 26.8± ±19.86* # 0.78±0.240 Cisap+ALBF( ) 165.4±13.64* 39.4±1.20* 104±4.98* # 0.44± One way F ANOVA P Values are mean ± SEM, n=5 in each group * P< compared to respective control (ANOVA followed by Dunnett test) # P< compared to F (Student t test) Discussion Nootropic agents have selective facilitatory effect on integrative functions of the central nervous system particularly on intellectual performance, learning capacity and memory (19). The present study indicates that the bark of Albizzia lebbeck, containing saponins possessed nootropic activity as indicated by shortening of transfer latency (TL). Both piracetam and ALBF met a major criterion for nootropic activity, namely improvement of memory in absence of cognitive deficit (20). Piracetam showed memory enhancing effect as it shortened the transfer latency on plus maze (21). ALBF improved memory retention 24 hour and one week as observed by increase in inflexion ratio. Similar results were obtained in object recognition test and passive shock avoidance test. 897

8 Different authors have reported that 5-HT 1A agonist impairs learning and memory. Earlier study found buspirone (partial 5-HT 1A agonist) not affecting learning and memory while recent studies have reported that buspirone disrupts such processes (22). In the present study, it was noted that administration of buspirone along with ALBF impairs learning on day 2 and day 9 as indicated by decrease in inflexion ratio on day 2 and day 9. In object recognition test, buspirone decreased Discrimination index as compared to ALBF treated group. In passive shock avoidance test also, latency to reach SFZ and mistakes in 15 min. were increased indicating buspirone impaired acquisition and retention. Menses et al (23) suggested that both 5-HT 1B and 5-HT 2A/2C receptor play a significant role in consolidation of the learning. It has been reported that m-cpp produced deficit in spatial learning while ketanserine and ritanserine improved the consolidation of learning in spatial discrimination. The effects of m-cpp most probably were mediated via 5-HT 2C receptor and kitanserine improved learning possibly via 5-HT 2A receptor. All these findings suggest that 5- HT 1B and 5-HT 2A/2C receptors are involved in learning and memory. In present study Mianserin and Cyproheptadine along with ALBF showed impairment of learning and memory on day 2 and day 9 as shown by decrease in inflexion ratio on day 2 and 9 and increase in latency to reach SFZ in passive shock avoidance test. The ability of 5-HT 3 receptor antagonists to facilitate the cholinergic transmission in cortical brain tissue raised the possibility that these compounds may be effective in treatment of cholinergic dysfunction in aging, alzheimer s disease and other functionally similar disorders.5-ht 3 antagonists inhibited the impairment in performance caused by cholinergic deficits in passive shock avoidance task in mice (24). In the present study, ondansetron along with ALBF enhanced learning and memory as shown by increase in inflexion ratio on day 2 and day 9 in elevated plus maze test, decrease in latency to reach SFZ and mistakes made in 15 min. in passive shock avoidance test and increase in Discrimination index in object recognition test. 5-HT 4 receptor appeared to be involved in the regulation of cognitive processes in mice. Administration of 5-HT 4 receptor antagonist (SDZ , GR ) provoked amnesia in passive shock avoidance test and 5-HT 4 receptor agonist (BIMU1, BIMU8) are able to prevent amnesia induced not only by 5-HT 4 antagonists but also by anti muscarinic agent and exposure to hypoxic environment. SL (5-(8-amino-7-chloro-2, 3-dihydro-1, 4- benzodioxin-5-yl)-3-[1-(2-phenylethyl)-4-piperidinyl)-1, 3, 4-oxadiazol-2(3H)-one monohydrochloride) is a novel benzodioxanoxadiazolone compound with high affinity for human 5-HT 4 receptors and good selectivity. SL potently improved performance in several tests of learning and memory. In the object recognition task, it improved retention at 24 h when administered i.p. or p.o. This effect was antagonized by the 5-HT 4 antagonist SDZ 205,557, demonstrating that the promnesic effects of SL are mediated by 5-HT 4 agonism. SL also reversed the cognitive deficits of aged rats in the linear maze task and the scopolamine-induced deficit of mice in the water maze task (25). In present study, cisapride along with ALBF showed enhancement of learning and memory on day 2 and 9 and also improved performance in passive shock avoidance test. In object recognition test, it restored object discrimination. Fluoxetine, zimelidine, sertraline, paroxetine, fluvo-xamine, indalpine and citalopram are the selective inhibitors of the serotonin reuptake (SSRIs) that have been most widely studied for their effects on learning and memory. By blocking the membrane uptake carrier, which transports serotonin from the extracellular space to inside the 5-HT nerve terminals, these compounds raise synaptic concentrations of serotonin and presumed to increase serotonergic transmission Surprisingly, in most instances these studies showed that treatment with SSRIs improved performance on learning tasks in animals and humans. However, inconsistent and contrary results do exist (26). 898

9 In the present study, fluoxetine administered along with ALBF enhanced learning and memory as shown by increase in inflexion ratio on day 2 and day 9 in elevated plus maze test, decrease in latency to reach SFZ and mistakes made in 15 min. in passive shock avoidance test and increase in Discrimination index in object recognition test. Thus it is concluded that 5-HT 3 and 5-HT 4 receptors improve learning and memory probably by facilitating cholinergic transmission. Acknowledgments The authors would like to thank Dr V. S. Kasture and Dr. S. C. Pal for their help in the correct identification of plant and its extraction and also for their support and encouragement. References 1. Lee SC, Moon YS, You KH. Effect of red ginseng saponins and nootropic drugs on impaired acquisition of ethanol-treated rats in passive avoidance performance. J. Ethnopharmacol 2000; 69: Iyer MR, Pal SC, Kasture VS, Kasture SB. Effect of Lowsonia inermis on memory and behavior mediated via monoamine neurotransmitters. Indian J Pharmacol 1998; 30: Brahmankar S, Kasture V, Kasture S. Nootropic and behavioural actions of saponins isolated from bark of Albizia lebbeck.journal of natural remedies 2001; 1(2): Rai KS, Rao MS, Karanth KS, Murthy KD. Clitoria ternatea enhances learning and memory An experimental study on rats. Indian Journal of Pharmacology 2000; 32: Singh HK, Dhawan BN. Neuropsychopharmacological effects of the Ayurvedic Nootropic Bacopa monnieri Linn. (Bramhi). Indian J Pharmacol 1997; 29: Jaiswal AK, Bhattacharya SK. Effect of Shilajit on memory anxiety and brain monoamines in rats. Indian J Pharmacol 1992; 24: Ying Y, Zang JT, Shi CZ, Liu Y. Study on the nootropic mechanism of ginsenoside Rb1 and Rb1-influene on mouse brain development. Acta Pharm Sin 1994; 29: Pal BC, Achari B, Yoshikawa K, Arihara S. Saponins from Albizzia lebbeck. phytochemistry 1995; 38: Veda M., Tokunaga T, Okazaki M, Sata NU. Albizziahexoside, ahexaglycosylated saponin isolated from leaves of Albizzia lebbeck. Natural Product Research 2003; 17(5): Thenmozhi V, Elango V, Sadique. Anti-inflammatory activity of some Indian medicinal plants. Journal of Ancient Science of life 1989; 8[3&4]: Kasture VS, Kasture SB, Pal SC. Anticonvulsant activity of Albizzia lebbeck leaves. Indian J Exp Biol 1996;34(1): Besra SE, Gomes A, Chaudhury L, Vedasiromoni JR, Ganguly DK. Antidiarrhoeal activity of seed extract of Albizzia lebbeck. Indian Journal of pharmacology 1999; 31(1): Haque N, Chowdhury SA, Nutan MT, et al.evaluation of anti tumour activity of some medicinal plants of Bangladesh by potato disc bioassay. Fitoterapia 2000;11(5):

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