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Investigations of Investigations of Sapindus trifoliatus Sapindus

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					       Research
        Paper

                                                   dopaminergic
         Investigations of Sapindus trifoliatus in dopaminergic and
          serotonergic
          serotonergic systems: Putative antimigraine mechanisms
                           D. K. Arulmozhi*, **, A. Veeranjaneyulu*, S.L. Bodhankar**, S.K. Arora*



                                                   ABSTRACT
          *New Chemical Entity                     Objective: To evaluate the potential dopaminergic and serotonergic receptor-mediated modu-
 Research, Lupin Research Park,
                                                   latory effect of the aqueous extract of Sapindus trifoliatus [(ST), (family: Sapindaceae)], a
   Village Nande, Taluk Mulshi,
                 Pune-411 042.                     traditional phytomedicine used in the treatment of hemicrania (migraine), using animal mod-
              ** Department of                     els and receptor assays.
                 Pharmacology,                     Materials and Methods: ST (at 20 and 100 mg/kg, i.p. doses) was evaluated for its effect on
            Bharati Vidyapeeth,                    apomorphine-induced climbing behavior, 5-hydroxytryptophan (l-5-HTP)-induced serotonin
    Poona College of Pharmacy,                     syndrome, and MK-801-induced hyperactivity in mice. The radioligand binding studies for
           Pune-411 038. India.                    various receptors and enzymes were carried out (outsourced) using standard procedures at
                                                   250 μg/ml concentration of ST.
              Received: 1.9.2004                   Results: ST significantly inhibited the apomorphine-induced climbing behavior, the l-5-HTP-
              Revised: 3.12.2004
                                                   induced serotonin syndrome and MK-801-induced hyperactivity in mice. In the receptor
             Accepted: 8.12.2004
                                                   radioligand binding studies, ST exhibited affinity towards dopamine D2, 5-HT2A receptors.
               Correspondence to:                  Conclusion: The results of the behavioral studies in mice indicate that ST modulated D2 and
                A. Veeranjaneyulu                  5-HT 2A receptor-mediated paradigms. The radioligand binding studies supported these
                           Email:                  observations, suggesting the possible involvement of dopaminergic and serotonergic
      addepalli@lupinpharma.com;                   mechanisms in the antimigraine activity of ST.
             add_bits@yahoo.com
                                                   KEY WORDS: Dopamine, headache, serotonin


Introduction                                                                     contraindicated in coronary artery disease. Hence, there is a
                                                                                 real need for better prophylactic/abortive agents. At present
    Migraine is characterized by attacks of intense pulsatile                    the mode of action of prophylactic treatment is mostly un-
and throbbing headache, typically unilateral in nature. A mi-                    known.[6]
nority of patients experience a more specific sensory distur-                        Although most recent studies have focused on the role of
bance prior to the headache, called 'aura' which usually has                     serotonin in migraine, the dopaminergic system is also impli-
visual disturbances. Migraine affects a substantial fraction (10-                cated in the pathogenesis of migraine.[7] Clinical and pharma-
20 %) of world population (more women than men). Attacks                         cological evidence suggests that hypersensitivity of the
are episodic and, by their very nature resolve with time. Asso-                  dopaminergic system could be involved in the pathogenesis of
ciated symptoms, such as nausea, vomiting and heightened                         migraine.[8]
sensitivity to light (photophobia), and sound (phonophobia) may                      Phytomedicine has offered an alternative source of therapy
occur during the headache phase. The headache phase may be                       for migraine sufferers, and provided some additional infor-
preceded by symptoms such as mood fluctuations and                               mation about the pathogenesis of migraine. [9,10] Feverfew
gastrointestinal disturbances.[1]                                                (Tanacetam parthenium) and butter-bur (Petasites hybridus)
    The diagnosis of migraine remains subjective since it re-                    are some of the plants that have been used for centuries for
lies on self-reports of various symptoms.[2] Although a wide                     relief of migraine.[11,12]
variety of antimigraine agents such as antiinflammatory drugs,                       Sapindus trifoliatus (ST) Linn. family Sapindaceae is a
ergots, 5-HT1 receptor agonists (triptans), 5-HT2 receptor an-                   medium-sized deciduous tree largely found in south India. The
tagonists and antiemetics[3-5] are available, there are no objec-                pericarp has a high content of saponins and sugars. The sa-
tive guidelines concerning the selection of a particular thera-                  ponin moiety is characterized as hederagenin group of
peutic approach in a given individual. The molecular patho-                      glycosides. The pericarp has been reported to possess vari-
physiology of migraine is not fully understood, thus making it                   ous medicinal properties. It is regarded as a tonic, stomachic,
very difficult to select the most efficacious therapeutic agent.                 spermicidal and useful in the treatment of hemicrania (mi-
Some of the available drugs have side effects and triptans are                   graine) etc.[13] A thick watery solution of the pericarp is used

120         Indian J Pharmacol   | April 2005 | Vol 37 | Issue 2 | 120-125
                                                                                       Mechanisms of the action of Sapindus trifoliatus


for the relief of hemicrania, hysteria or epilepsy.[13] However,       The method of Chung et al [16] was followed with minor
there have been no systematic pharmacological studies re-          modifications. Each mouse was placed in a cylindrical wire
ported on ST for its antimigraine use.                             mesh cage (height 13 cm, diameter 14 cm and mesh size 3
    Since ST is used in folklore medicine in the treatment of      mm) for 1 h prior to the experiments. Saline (10 ml/kg, i.p.) or
hemicrania, an investigation was undertaken to explore the         ST (20 or 100 mg/kg, i.p.) or haloperidol (1 mg/kg, i.p.) was
possible mechanism. In the present study, the lyophilized aque-    administered 30 min prior to apomorphine (2.5 mg/kg, i.p.).
ous extract of ST was used in animal studies to predict            Climbing behavior was assessed at 5-min intervals up to 20
dopamine and serotonin antagonist activities; along with in        min, starting 10 min after apomorphine, using the following
vitro binding affinity studies in various receptors.               scoring system. 0-no paws on the cage, 1-two paws on the
Materials and Methods                                              cage, 2-four paws on the cage. The score recorded for each
                                                                   animal was based on the position of the animal at the moment
Drugs
                                                                   it was first observed. The total time spent on the cage was
    Apomorphine, pargyline, haloperidol and l-5-HTP were
                                                                   also recorded for each animal. An observer unaware of the
obtained from Sigma Chemical Co (ST. Louis, USA). MK-801
                                                                   specific treatments recorded the observations.
was obtained from RBI (USA) and methysergide was procured
from Tocris (Avonmouth, UK). Except haloperidol, which was
                                                                   l-5-HTP-induced serotonin syndrome
dissolved in 0.01% lactic acid solution, all other drugs and
                                                                       The syndrome was measured after placing a mouse in a
chemicals were dissolved in normal saline. All the drugs were
                                                                   Perspex cage for a 30-min habituation period. The animal was
administered intraperitoneally.
                                                                   then injected with pargyline (75 mg/kg, i.p.) in order to pre-
Plant material and extraction procedure                            vent the rapid degradation of l-5-HTP Thirty min later, ST (20
                                                                                                          .
    Pharmacognostically identified dried pericarp of fruits of     or 100 mg/kg, i.p.) or methysergide (10 mg/kg, i.p.) was ad-
Sapindus trifoliatus Linn, family Sapindaceae were collected       ministered. After 30 min the animal received l-5-HTP (50 mg/
from the local market. Aqueous extract of ST was prepared as       kg, i.p.) and was returned to test cage. After 20 min, the sero-
reported.[14] Briefly, 100 grams of the pericarp was soaked in     tonin syndrome was assessed every 10 min through 50 min.
400 ml of distilled water for 16 h. The percolate was then de-     Five behavioral parameters, such as tremor, hind limb exten-
canted, centrifuged and filtered through Whatman (No.1) fil-       sion, forepaw treading, head weaving and head twitch were
ter paper to obtain a clear extract (300 ml). The extraction       monitored using the following scoring system: 0-absent, 1-
process was repeated again with the same volume of distilled       moderate, 2-marked.[16, 17] A trained observer unaware of the
water. The percolates were pooled and lyophilized to give a        specific treatments recorded the findings.
brown colored powder (68% yield). Acid hydrolysis of the ex-
tract yielded only one glycone, which was identified as            MK-801-induced hyperactivity
hederagenin. Therefore, estimation of the saponins present in          Mouse locomotor activity was measured using a compu-
the extract was calculated as hederagenin. The content of          terized animal activity meter (Opto Varimex Auto Track Sys-
hederagenin in the extract was estimated by boiling it with        tem, Columbus Instruments, OH, USA), made of clear perspex
50% methanolic hydrochloric acid. The entire mixture was           box (40 x 40 x 40 cm). An array of 15 infrared emitters (spaced
evaporated to dryness and reconstituted in methanol. HPLC          at 2.65 cm intervals; beam wave length 940 nm) measured
estimation was carried out using Kromasil C-18 column (5           the animal’s activity along the single axis of motion. To mini-
μm, 250 x 4.6 mm) with gradient elution (0.1% formic acid          mize the variation among the individuals, mice were placed in
and acetonitrile in the ratio of 80: 20 v/v) and evaporative       pairs in the locomotor activity boxes for a 30 min period. Af-
light-scattering detector. The concentration of hederagenin was    ter this time, the animals were injected with ST (20 or 100
found to be between 5.61-6.97 % by weight of the extract.[15]      mg/kg, i.p.) or haloperidol (0.1 mg/kg, i.p.) or vehicle and then
Animals                                                            returned to the test boxes. The recording was begun immedi-
    Adult male Swiss albino mice (18-22 g) were obtained from      ately. After a 30 min period, the animals were injected with
the Research Animal Facility of Poona College of Pharmacy          MK-801 (0.3 mg/kg, i.p.) and the recording was continued
(PCP), Pune, India. On arrival, the animals were placed at ran-    immediately. Activity was measured for a further 90 min. The
dom and allocated to treatment groups (10 animals per treat-       perspex boxes allowed for continuous visual monitoring of the
ment) in polypropylene cages with paddy husk bedding. They         animals in addition to the automated measurement of loco-
were housed at a temperature of 24+2 oC and relative humid-        motor activity. The photocell counts that were measured every
ity of 30-70% with a 12:12 light:dark cycle. All had free access   minute were grouped into 10 min time intervals.[16, 18]
to water filtered through Aquaguard® and standard pelleted
laboratory animal diet. The experimental procedures and            Receptor radioligand binding studies
protocols used in the present study were reviewed and ap-              The receptor radioligand binding studies were carried out
proved by the Institutional Animal Care and Use Committee of       by NovaScreen Biosciences Corporation, USA. The studies were
PCP Pune, India, and were in accordance with the guidelines
    ,                                                              done as per the standard protocols[19-21] and the details of the
of the Committee for the Purpose of Control and Supervision        receptor source ligands and reference compounds used are
of Experiments on Animals (CPCSEA), Ministry of Forests and        listed in Table 1.
Environment, Government of India. Experiments were carried
out between 1000 and 1700 h.                                       Statistical analysis
Apomorphine-induced climbing behavior                                 Results are expressed as mean+SEM. Comparisons be-

                                                                            Indian J Pharmacol   | April 2005 | Vol 37 | Issue 2 | 120-125   121
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | April 2005 | Vol 37 | Issue 2 | 120-125   Indian J Pharmacol         122
headache, a finding that generated considerable scientific in-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  evant targets in migraine (Table 1) at an optimal concentra-
soon proven to be beneficial in the treatment of acute migraine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 affinity towards various receptors and enzymes that are rel-
ing contraction of dog/rabbit saphenous vein. Sumatriptan was                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       In receptor radioligand-binding studies, ST exhibited an
identified as a selective agonist at 5-HT1-like receptor mediat-
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Discussion
    The antimigraine gold standard sumatriptan was originally
tion of 250 μg/ml.[21]                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          are shown in Table 1.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    The radioligand binding data of ST on selected receptors
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Receptor ligand binding studies
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                cantly (P<0.001) inhibited the MK-801-induced hyperactivity.
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        1                                           ST (20 and 100 mg/kg) and haloperidol (0.1 mg/kg) signifi-
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                MK-801-induced hyperactivity
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                l-5-HTP-induced head twitches (P<0.05) (Figure 3).
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                ST 20 mg/kg and methysergide (10 mg/kg, i.p.) inhibited only
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                the five behavioral syndromes induced by l-5-HTP however
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    *                                                                                                                                                                                                                                                                   6                                           ST (100 mg/kg, i.p.) significantly (P<0.001) reduced all
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                l-5-HTP-induced behavioral syndrome
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               Cumulative climbing score




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        7
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                climbing behavior in mice (Figures 1 and 2).
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                significantly (P<0.05-0.001) inhibited apomorphine-induced
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    ST (20 and 100 mg/kg, i.p.) and haloperidol (1 mg/kg, i.p.)
           **P<0.001.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           Apomorphine-induced climbing
           treatment. A score of 10 is the maximum possible. *P<0.05,
           intervals for 20 min, starting 10 min after apomorphine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          Results
           climbing score for groups of ten mice assessed at 5-min
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                suitability. A P value of <0.05 was considered as significant.
           kg, i.p.). Each column represents mean+SEM of total
           behavior (climbing score) induced by apomorphine (2.5 mg/                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            of variance (ANOVA) followed by Dunnetts’ post test as per
Figure 1. The effect of Sapindus trifoliatus and haloperidol on climbing                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        tween the groups were made by Student’s ‘t ’ test or analysis
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  *The receptor radioligand binding studies were carried out by NovaScreen Biosciences Corporation, USA .
                                                                                                                                                                                                                                             84.10                                                                                                                                                                                                                                                                                                                                                                                                                                                                         L-Arginine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         [3H]Arginine                                      Rat cerebellum          NOS (Constitutive-neuronal)
                                                                                                                                                                                                                                             15.06                                                                                                                                                                                                                                                                                                                                                                                                                                                                         Glibenclamide                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      [3H]Glibenclamide                                 Rat cortex              Potassium Channel, ATP-sensitive
                                                                                                                                                                                                                                             2.94                                                                                                                                                                                                                                                                                                                                                                                                                                                                          (+)-MK-801 HMaleate                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                [3H]TCP                                           Rat forebrain           Glutamate, NMDA
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        (Dihydropyridine site)
                                                                                                                                                                                    33.93                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Nifedipine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      [3H]Nitrendipine                                 Rat cortex                      Calcium channel L type
                                                                                                                                                                                    17.40                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Mianserin HCl                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   [3H]Mesulergine                                  Porcine choroid plexus          Serotonin, 5HT2C
                                                                                                                                                                                    47.53                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Methysergide maleate                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            [3H]Ketanserin                                   Rat cortex                      Serotonin, 5HT2A
                                                                                                                                                                                    3.17                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               5-Carboxytryptamine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             [3H]-5-CT                                        Human cortex                    Serotonin, 5HT1D (Human)
                                                                                                                                                                                    41.71                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Serotonin                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       [125I](-)-Cyanopindol, iodo                      Rat striata                     Serotonin, 5HT1B
                                                                                                                                                                                    13.41                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              (+/-)-8-OH-DPAT HBr                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             [3H]-8-OH-PAT                                    Bovine striata                  Serotonin, 5HT1A
                                                                                                                                                                                    13.38                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Imipramine HCl                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  [3H]Citalopram, N-Methyl                         Rat forebrain                   Serotonin transporter
                                                                                                                                                                                    13.69                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Naloxone HCl                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    [3H]Naloxone                                     Rat forebrain                   Opiate, Non -selective
                                                                                                                                                                                    7.14                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               N-a-Methylhistamine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             [3H]N-a-MeHistamine                              Rat forebrain                   Histamine, H3
                                                                                                                                                                                    27.52                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Tiotidine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       [125I]-Aminopotentidine                          Guinea pig spleen               Histamine, H2
                                                                                                                                                                                    0.25                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               Triprolidine HCl                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                [3H]Pyrilamine                                   Bovine cerebellum               Histamine, H1
                                                                                                                                                                                    55.18                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              (+/-)-7-OH-DPAT HBr                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             [3H]7-OH-DPAT                                    Rat cDNA/SF9                    Dopamine, D3 recombinant
                                                                                                                                                                                    104.54                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             (+/-)Sulpiride                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  [3H]Sulpiride                                    Rat striata                     Dopamine, D2
                                                                                                                                                                                    20.78                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              R(+)-SCH 23390 HCl                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              [3H]SCH 23390                                    Rat striata                     Dopamine, D1
                                                                                                                                                                                    49.38                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Phentolamine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    [3H]RX 821002                                    Rat cortex                      Adrenergic, alpha-2, Non-selective
                                                                                                                                                                                    58.96                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Phentolamine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    [3H]7-MeOxy-Prazosin                             Rat forebrain                   Adrenergic, alpha-1, Non-selective
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    3
                                                                                                                                                                                    10.76                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Neca                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            [ H] NECA                                        Bovine striatum                 Adenosine, Non-selective
  % inhibition at 250 μg/ml                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         Reference compound                                                                                                                                                                                                                                                                                                                                                                                                                                                                     Ligand             Receptor source                                              Receptor
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Selected receptor radioligand binding data of ST
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            Table 1
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             Arulmozhi DK, et al.
123                                                                                                        120 125                                                                                                                ssue 2                                                                                                                               Vo 37                                                                                                     2005                                                                                                                  Ap                                                                                                                           nd an J Pha maco
metoclopromide.[32]                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       The locomotor stimulant effects of MK-801 (a non-com-
ten controlled with dopamine receptor antagonist                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      response.[25]
teers.[31] Migraine associated with nausea and vomiting is of-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        HTP-induced behavioral syndromes, especially the head twitch
agonist administration, when compared with normal volun-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              (5HT2) antagonist, reportedly inhibited the components of l-5-
cidence of dopaminergic symptoms following acute dopamine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 The prophylactic antimigraine methysergide, a serotonin
activation. Furthermore, migraine patients show a higher in-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          this model.[24]
accompanying symptoms could be related to dopaminergic                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                and D1 and D2 antagonists were proved to be efficacious in
involvement of dopamine in migraine and most prodromal and                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            mediated through the activation of both D1 and D2 receptors,[23]
    Clinical and pharmacological evidence suggests that the                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               Apomorphine-induced climbing response is reported to be
of 5-HT-induced syndromes (Figure 3).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 (5HT2A) (47.53%) receptors.
lated to the higher dose required for the significant inhibition                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      hibited affinity towards dopamine D2 (104.54%) and serotonin
D2 receptors in the ligand binding studies, which may be re-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          ity with the ligand binding studies, where ST (250 μg/ml) ex-
hibited less affinity towards 5-HT2A receptors than dopamine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          in the central nervous system. The current data is in conform-
rotonin agonists in the aforementioned animal models. ST ex-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          actions mediated through dopamine and serotonin receptors
inhibiting the behavioral effects induced by dopamine and se-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             In the present investigation, ST exhibited pharmacological
exhibited dopamine D2 and serotonin (5HT2) antagonism by                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              mediation were studied in the present investigation.
    ST, a phytomedicine used for the treatment of hemicrania,                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         ble effects of ST through 5-HT2A and dopamine D2 receptor
stimulant effect.[27]                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 adrenoceptors was ruled out in functional studies, the possi-
monoaminergic neurotransmission underlies the locomotor                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               hemicrania in folklore medicine and the role of 5-HT1B and α-
serotonin release in brain favors the notion that increased                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           respectively.[22] Since ST has been used in the treatment of
mate.[29,30] The ability of MK-801 to increase dopamine and                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           and 5-HT1B receptors in rabbit aorta and rabbit saphenous vein
regulation of forebrain dopaminergic systems via gluta-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               affinity towards acute migraine targets viz. α-adrenoceptors
through 5-HT2A receptors could play a permissive role in the                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          in rabbit saphenous vein.[22] However, ST failed to exhibit an
NMDA antagonists.[28] The serotonergic system modulated                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               studied for its potential affinities towards 5-HT1B/1D receptors
volved in the alteration of serotonin turnover produced by                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            vascular smooth muscles. In line with the above, ST has been
tions, dopaminergic-serotonergic interactions might be in-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            evidence that the 5-HT1B receptor mediates the contraction of
tum. In addition to direct glutamatergic-serotonergic interac-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        human cerebral arteries. Indeed, now there is overwhelming
cortex, hippocampus, nucleus accumbens, amygdala and stria-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           receptor mediating cerebral vasoconstriction in the cat and
creases 5-HT turnover in several brain regions including the                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          larities between the 5-HT1B and/or 5-HT1D receptors and 5-HT1
pharmacological data have demonstrated that MK-801 in-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                it was pointed out that there were strong pharmacological simi-
antagonize MK-801-induced hyperactivity in mice.[18] Recent                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           receptor causing contraction of the dog/rabbit saphenous vein,
Dopamine (5HT2) and serotonin antagonists are reported to                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             smooth muscle. While it was reported to act on the 5-HT1-like
creased dopamine and serotonin release in the brain.[26, 27]                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          receptor involved in the contraction of the cerebrovascular
petitive NMDA channel blocker) have been correlated to in-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            terest towards the identification of the selective serotonin



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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    do                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     ST




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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        m                                                                                                                                                                                                                                                                                                                                                           m                                                                                                                                                                                                                                                                                                                           m
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                                                                                                                                                     000000000000                                                                                                                   00000000000000                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                000000000000
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                                                                                                                                        00 00 00 00 00 00 00 00 00 00 00 00                                                                                           00 00 00 00 00 00 00 00 00 00 00 00 00 00                                                                               00 00 00 00 00 00 00 00 00 00 00 00                                                                            00 00 00 00 00 00 00 00 00 00 00 00                                                                                  00 00 00 00 00 00 00 00 00 00 00 00 00 00                                                                                        00 00 00 00 00 00 00 00 00 00 00 00                                                                 00 00 00 00 00 00 00 00 00 00 00 00                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00
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                                                                                                                                                                                                                           00                                                                                                                                    00                                                                                                         0                                                                                                                   00                                                                                                                           00                                                                                                        00                                                                                                   00                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     0000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000
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                                                                                                                                           000 000 000 000 000 000 000 000 000 000 000 000                                                                               000 000 000 000 000 000 000 000 000 000 000 000 000 00                                                                 000 000 000 000 000 000 000 000 000 000 000 000                                                                 000 000 000 000 000 000 000 000 000 000 000 000                                                                      000 000 000 000 000 000 000 000 000 000 000 000 000 00                                                                           000 000 000 000 000 000 000 000 000 000 000 000                                                     000 000 000 000 000 000 000 000 000 000 000 000                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        00
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                                                                                                                                                                                                                                                                                                                                                                  0                                                                                                                                                                                                                                                                                                                                                           0
                                                                                                                                             00 00 00 00 00 00 00 00 00 00 00 00                                                                                           00 00 00 00 00 00 00 00 00 00 00 00 00 0                                                                               00 00 00 00 00 00 00 00 00 00 00 00                                                                             00 00 00 00 00 00 00 00 00 00 00 00                                                                                  00 00 00 00 00 00 00 00 00 00 00 00 00 0                                                                                         00 00 00 00 00 00 00 00 00 00 00 00                                                                 00 00 00 00 00 00 00 00 00 00 00 00                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00
                                                                                                                                              000000000000                                                                                                                                                                                                                                         000000000000                                                                                                    000000000000                                                                                                                                                                                                                                          000000000000                                                                                        000000000000
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                                                                                                                                              00 00 00 00 00 00 00 00 00 00 00 00                                                                                           00 00 00 00 00 00 00 00 00 00 00 00 00 00                                                                              00 00 00 00 00 00 00 00 00 00 00 00                                                                             00 00 00 00 00 00 00 00 00 00 00 00                                                                                  00 00 00 00 00 00 00 00 00 00 00 00 00 00                                                        00 00 00 00 00 00 00 00 00 00 00 00                                                                                                 00 00 00 00 00 00 00 00 00 00 00 00                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00
                                                                                                                                               000000000000000000000000000000000000000000000000000000000000                                                                  00000000000000000000000000000000000000000000000000000000000000000000                                                   000000000000000000000000000000000000000000000                                                                   000000000000000000000000000000000000000000000000000000000000                                                         00000000000000000000000000000000000000000000000000000000000000000000                                                                                                                                                                 000000000000000000000000000000000000000000000000000000000000                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 0
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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         To a score
                                                                                                                                                    00 00 00 00 00 00 00 00 00 00 00 00                                                                                           00 00 00 00 00 00 00 00 00 00 00 00 00 00                                                                             00 00 00 00 00 00 00 00 00 00 00 00                                                                              00 00 00 00 00 00 00 00 00 00 00 00                                                                                  00 00 00 00 00 00 00 00 00 00 00 00 00 00                                                                                                                                                     00 00 00 00 00 00 00 00 00 00 00 00
                                                                                                              000000000000                                                                                                                                                         00 00 00 00 00 00 00 00 00 00 00 00 00 00                                                                             00 00 00 00 00 00 00 00 00 00 00 00                                                                              00 00 00 00 00 00 00 00 00 00 00 00                                                                                  00 00 00 00 00 00 00 00 00 00 00 00 00 00
                                                                                                                                                                                                                                                                                    00000000000000                                                                                                        000000000000                                                                                                     000000000000                                                                                                         00000000000000
                                                                                                                                                                                                                                                                                                                                                                                                          000000000000                                                                                                     000000000000

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               *
                                                                                                                                                                                                                                                                                    0 0 0 0 0 0 0 0 0 0 0 0 0 00                                                                                                                                                                                                                                                                                                                                0 0 0 0 0 0 0 0 0 0 0 0 0 00
                                                                                                                                                                                                                                           00 00 00 00 00 00 00 00 00 00 00 00 00 0                                                                                                                       00 00 00 00 00 00 00 00 00 00 00 00                                                                              00 00 00 00 00 00 00 00 00 00 00 00                                                                                  00 00 00 00 00 00 00 00 00 00 00 00 00 0

                                                                                                      *
                                                                                                                                                                                                                                                                                                                                                                                                           00 00 00 00 00 00 00 00 00 00 00 00                                                                              00 00 00 00 00 00 00 00 00 00 00 00                                                                                  00 00 00 00 00 00 00 00 00 00 00 00 00 00
                                                                                                                                                                                                                                                                                                                                                                         000000000000000000000000000000000000000000000000                                                                                                                                                                         00                                                                                                                           00

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     6
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             0000000000000000000000000000000000000000000000000000000000                                                           0000000000000000000000000000000000000000000000000000000000000000000
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               000 000 000 000 000 000 000 000 000 000 000 000                                                                      00000000000000
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    00 00 00 00 00 00 00 00 00 00 00 00 00 00
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 00 00 00 00 00 00 00 00 00 00 00 00                                                                                 00 00 00 00 00 00 00 00 00 00 00 00 00 0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   00 00 00 00 00 00 00 00 00 00 00 00                                                                                  00000000000000                                                                                                                                                                                                                                                                                                                               7
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               Cumulative climbing duration (min)




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          20
                                                                                                                                                                                                                                      Me hyse g de 10 mg
                                                                                                     ST 20 mg                                                                                                                         Sa ne        ST 100 mg
          compared to saline.
          the maximum possible. *P<0.05, **P<0.001 when                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         treatment. *P<0.05, **P<0.001 when compared to saline.
          min, starting 20 min after l-5-HTP treatment. A score of 8 is                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         assessed for 20 min, starting 10 min after apomorphine
          groups of ten mice assessed at 10 min intervals for 30                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                time (min) spent on the cage for groups of ten mice
          Each column represents mean+SEM of total score for                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    mg/kg, i.p.). Each column represents mean+SEM of total
          serotonin syndrome induced by l-5-HTP (50 mg/kg, i.p.).                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               behavior (climbing duration) induced by apomorphine (2.5
Figure 3. The effect of Sapindus trifoliatus and methysergide on                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      Figure 2. The effect of Sapindus trifoliatus and haloperidol on climbing
o a us                                                                                                    Mechan sms o he ac on o Sap ndus
  Arulmozhi DK, et al.


  Figure 4. The effect of Sapindus trifoliatus and haloperidol on                                                                           teers and migraine sufferers.[38] Furthermore, we have recently
            hyperactivity induced by MK-801 (0.3 mg/kg, i.p.). Each                                                                         reported the antinociceptive activity of ST in nitroglycerin-in-
            point represents mean+SEM of number of light beam
                                                                                                                                            duced hyperalgesic rats.[14] In a double-blind study, selective
            interruptions for groups of ten pairs of mice. #P<0.05,
            ##
               P<0.001 compared to vehicle/vehicle, *P<0.001                                                                                serotonin (5HT2) receptor antagonist ritanserin was highly ef-
            compared to vehicle/MK-801 group.                                                                                               fective in reducing Pain Total Index and analgesic consump-
                                                                                                                                            tion in chronic headache.[39]
                            Ve hic le /S T/Ha lope ridol                                                                                        In view of the modulatory role of ST on dopamine D2 and
                                                                                                                                            serotonin (5HT2) receptors, there is a strong possibility of the
                            6000
                                                                                  ##
                                                                                                                                            involvement of these receptors in its antimigraine activity. The
                                                  V ehic le/M K-8 0 1 ##
                                                                                                                                            present data adds value to the reported ethnomedical usage
Ambulatory counts /10 min




                                                                  ##                     ##   ##
                                                                                                                                            of ST in the treatment of hemicrania.
                                                                                                    ##
                                                                                                         ##
                            4000
                                                                                                                                            Acknowledgements
                                                             #                                                                              We wish to thank Dr. Vikas Kumar and Dr. N. Sridhar for critical comments on the
                                                                                                               Vehicle/Vehicle
                                                                                                                                            manuscript. Thanks also to Dr. V. Srivastava and Mr. S. K. Joshi for their constant
                            2000                                                                               Vehicle/MK-801               help in the extraction of ST. We hereby acknowledge NovaScreen Biosciences
                                                                                                               ST 20 mg/MK-801*
                                                                                                                                            Corporation, USA for carrying out the radioligand binding studies.

                                                                                                               ST 100 mg/MK-801*            References
                                 0
                                                                                                               Haloperidol 0.1 mg/MK-801*
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                                                                                                              Mechanisms of the action of Sapindus trifoliatus


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                                               3rd NORTH ZONE CONFERENCE
                                            INDIAN PHARMACOLOGICAL SOCIETY

                               Theme                  :           Clinical Pharmacology
                               Date                   :           23rd April 2005
                               Venue                  :           Delhi Institute of Pharmaceutical Sciences
                                                                  & Reasearh (DIPSAR), Sector-3, Pushp Vihar,
                                                                  New Delhi-17. INDIA

         The conference will include invited lectures, free paper presentations and poster presentations.
         Last date for the submission of abstract (200 words)- 5th April 2005. Registration fee -
         Rs. 500/-
                                                   For further information, please contact:

          Prof. (Dr.) S.S.Agrawal                                                                                             Dr. N.R Bishwas
          Principal, DIPSAR                                                                                                Additional professor,
          Pushp Vihar, Sector –III, N. Delhi.                                                                                     Dr.R.P Centre,
          President,Indian Pharmacological Society                                                                         A.I.I.M.S. New Delhi
          Chairman                                                                                                        Organizing Secretory
          3-NZCOP                                                                                                                      3-NZCOP
          Telephone:01126563771                                                                                         Telephone:9818870826




                                                                                                  Indian J Pharmacol   | April 2005 | Vol 37 | Issue 2 | 120-125    125

				
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