PHYTOCHEMICAL EVALUATION & PHARMACOLOGICAL …

www.wjpps.com Vol 4, Issue 12, 2015.

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Farheen et al. World Journal of Pharmacy and Pharmaceutical Sciences

PHYTOCHEMICAL EVALUATION & PHARMACOLOGICAL

SCREENING OF ETHANOLIC BARK EXTRACT OF

DOLICHONDRONE FALCATA & ROOT EXTRACT OF

SCROPHULARIA HYPERICIFOLIA FOR

PSYCHOPHARMACOLOGICAL ACTIVITIES IN MICE.

Mehnoor Farheen*¹, Sumera Mahreen Khatoon², Asfia Kouser³

*¹M.Pharm (Ph.D.) Associate Professor, Department of Pharmacology, Shadan Women’s

College of Pharmacy, JNTU, Hyderabad, India

²,³Pharmacology, Shadan Women’s College of Pharmacy, JNTU, Hyderabad, India.

ABSTRACT

Psychopharmacology is concerned with drug effects that modify the

behaviour of animal organisms. This study assessed the antidepressant,

antianxiety, antipsychotic, antiepileptic, hypnotic and sedative

activities of ethanolic extract of Dolichondrone falcata and

Scrophularia hypericifolia in diseases induced mice. Extraction of the

ethanolic extract from bark of Dolichondrone falcata and roots of

Scrophularia hypericifolia was performed by maceration. 24 mice were

divided into six groups. Group I consisted of normal mice that were

given only sterile saline solution and served as control group. Group II

mice consisted of normal mice induced with disease either through

drug or physically. Group III, IV, V consisted of normal mice induced

with depression, anxiety, psychosis, epilepsy. Oral administration of

extract resulted in significant antidepressant, antianxiety,

antipsychotic, antiepileptic, hypnotic and sedative effect and muscle relaxant activity. Group

VI consisted of normal mice induced with depression, anxiety, psychosis, epilepsy and were

treated with standard drug. The effect produced by the extracts were closely similar to the

standard drugs. In conclusion, the present study indicates that the ethanolic extract of

Dolichondrone falcata and Scrophularia hyperictifolia appears to exhibit antidepressant,

antianxiety, antipsychotic, antiepileptic, hypnotic and sedative activity, muscle relaxant

activity in disease induced mice.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 5.210

Volume 4, Issue 12, 905-934 RReesseeaarrcchh Article ISSN 2278 – 4357

Article Received on

05 Oct 2015,

Revised on 29 Oct 2015,

Accepted on 22 Nov 2015

*Correspondence for

Author

Dr. Mehnoor Farheen

M.Pharm (Ph.D.) Associate

Professor, Department of

Pharmacology, Shadan

Women’s College of

Pharmacy, JNTU,

Hyderabad, India.


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KEYWORDS: elevated plus maze, forced swim test, haloperidol induced catalepsy, cerebral

edema, neuronal degeneration.

INTRODUCTION

In this study the extracts of parts of the plants Dolichondrone Falcata and Scrophularia

Hypericifolia was evaluated for their use in the management of diseases like anxiety,

depression, memory disorder and was evaluated for sedative and hypnotic property. From the

thorough study and investigation of the available literature of plants. Both the plants have

shown that they serve as an important sources of many therapeutically efficient chemicals

like flavonoids, organic acids like sugar and terpenes and various other constituents. The

ethanolic extract of bark of dolichondrone falcata and roots of scrophularia hypericifolia was

evaluated for various psychopharmacological disorders in mice. The disease was induced by

drug or through physical induction.

REVIEW OF DRUG UNDER STUDY

Scrophularia hypericifolia [1, 2]

Scrophularia hypericifolia belong to the genus Scrophularia, represented by over 300 species

worldwide in Saudi Arabia, about 5 species were described. Genus Scrophularia is rich in

iridoid glycosides, phenylpropanoids and flavonoids.

Dolichondrone falcata [3, 4]

Dolichondrone falcata is a small deciduous tree in the Bignoniaceae family. It

is endemic to India.

MATERIALS AND METHODS

Collection and preparation of extract

Dried bark extract of dolichondrone falcata and dried root extract of scrophularia

hypericifolia were collected. The plants were taxonomically identified and authenticated by

DR. K. MADHAVA CHETTY assistant professor of botany department of pharmacognosy,

Sri Venkateshwara University, Tirupati.

Preparation of Plant Extracts

The plant extract was prepared by maceration [5]

process. Standard screening test [6]

of the

extract was carried out for various plant constituents using standard procedures. Acute

http://en.wikipedia.org/wiki/Bignoniaceae

http://en.wikipedia.org/wiki/Endemism

http://en.wikipedia.org/wiki/India


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toxicity studies [7]

were performed for the extracts according to the acute toxic classic method

as per the method of Litchfield and Wilcoxon (1949).

Experimental animals

Mice (20 – 50 gms) of either sex housed in standard conditions of temperature (55+ 5%) or

light (12 hrs. light /dark cycles) were used. Animals were randomly selected for grouping. All

experiments were performed according to the forms of the ethical conditions. (CPSCSEA).

Histopathological studies [8]

The animals were euthanized using anesthetic ether and their brains were dissected out. The

isolated organ was sliced into 5mm pieces and fixed in neural formalin (10% solution) for at

least 3 days. This was followed by dehydration with isopropyl alcohol of increasing strength

(70%, 80%, and 90%) for 12 hours each and the final dehydration was done using absolute

alcohol with about three changes for 12 hours each. Clearing was done by using chloroform

with two changes for 15 to 20 minutes each. After clearing the object pieces were subjected

to paraffin infiltration in automatic tissue unit. The organ pieces were then dropped into

molten paraffin quickly and allowed to cool. The blocks were cut using microtome to get

sections of thickness of 5µ. The sections were applied with egg albumin then allowed to

remain in an oven at 600C for 1 hour. Paraffin melts and egg albumin denatures, thereby

fixing tissues to the slide. Staining with Eosin and hematoxylin stain.

Fig 1: isolated brain of mice

Elevated plus maze /elevated zero maze[9]

The Elevated plus-maze (EPM) was used to evaluate the anxiolytic effects of the plant

extract. Group I Mice received normal saline, group II received control drug , group III mice


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received control drug (levodopa) and bark extract of dolichondrone falcata (400mg/kg),

group IV mice received control drug (levodopa) and root extract of scrophularia hypericifolia

(200mg/kg), group V were received control drug and combination of both extracts

(dolichondrone falcata + scrophularia hypericifolia) and group VI were received control drug

and standard drug (diazepam 3mg/kg) and 60 min later, they were individually placed in the

center of the EPM for 5 min. The number of entries to the open and enclosed arms, and time

spent in the open arms, were recorded. Diazepam (3 mg/kg, i.p.) was used as positive control.

Fig 4: elevated plus maze test

Locomotor activity[10]

The locomotor activity can be easily studied with the help of actophotometer in a digital

actophotometer, continuous beam of light falls on the photoelectric cells. After 30 min of

administration of extracts and 1hr of i.p administration animals were individually placed in

actophotometer and the digital count as the number of line crossing by animal due to beam

interruption, were recorded for 10 minutes.

Fig 5: locomotor activity

Novelty-induced hypophagia[11]

Rodents encountering a desirable food in a novel environment will consume very limited

quantities after considerable investigation. Mice tend to avoid exploration of novel open


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environments, yet are motivated to approach and consume palatable food. This inhibition of

feeding behavior has been termed hypophagia and is robust in both rats and mice. Singly

housed mice are trained to consume the palatable food source by introducing it to them in

their home cage for 30 min daily over three consecutive days. Diluted condensed milk in

plastic serological pipettes (10 mL) with attached sippers and rubber stoppers are mounted to

the wire cage lid. On the fourth day mice are tested in the home cage condition. Record the

latency to the first lick and the total volume consumed in 5-min intervals across the 30-min

session. Note any mice that do not consume any condensed milk. They should be excluded

from further testing as they failed the training protocol. On day 5, position the pipette in the

wire lid of the novel cage and place the mouse into the novel cage environment. Record

latency and total volume consumed.

Forced swim test [12]

The FST was carried out on mice according to the method of Porsolt et al. mice are

individually forced to swim inside a vertical plexiglass cylinder (height 40 cm, diameter 18

cm, containing 15 cm of water at 25o C). Mice when placed first time in cylinder are initially

highly active, vigorously swimming in circles, trying to climb the wall or diving to the

bottom. After 2-3 min this activity begins to subside and gets interspersed with phases of

immobility or floating of increasing duration. After 5 -6 min immobility reaches a plateau

where the animals remain immobile for approximately 80% of time. After 15 min the animals

are removed and allowed to dry in heated (32o C) enclosure and returned to their home cages.

After 24 hours test drug and standard drugs are administered. Animals were forced to swim

for 6 min, and the time spent in immobility during the last 5 min of a 6 min observation

period was recorded manually by the competent observer.

Fig 6: forced swim test


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Tail suspension test [13]

Tail suspension test is a facile means of screening the antidepressant drugs. The immobility

displayed by mice, when subjected to unavoidable and inescapable stress, reflects behavioral

despair, which in turn may reflect depressive disorders in human. Clinically effective

antidepressants significantly reduce the immobility that mice display following active and

successful attempts to escape when suspended by tail. The total duration of immobility was

quantified during a test period of 5min. Mice were considered immobile when they were

completely remain motionless for at least 30s.

Fig 7: tail suspension test

Test for motor coordination (Rota-rod test) in mice [14]

It is one of the classical methods introduced by dunham and miya in 1957 for the evaluation

of drugs interfering with the motor co-ordination activity by testing their ability to remain on

a revolving rod. The apparatus consist of a horizontal wooden rod or metal rod coated with

rubber with 3cm diameter attached to a motor with the speed set at 2 rotations per minute.

Only those mice, which demonstrate their ability to remain on the revolving rod for at least

60s, are chosen for the test.

Fig 8: motor coordination test


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Pentobarbital-induced hypnotic test[15]

This test is indicative of CNS depressant activity of the test compound. Hypnotic, sedatives,

tranquilizers and even antidepressants have been shown to prolong the sleeping time after the

single dose of pentobarbital.

The loss of righting reflex is used as a criterion for the duration of sleep in this model. After

30 min of drug administration the animals are then placed on their back on thermo-

statistically warmed pad (37o C) and the duration of the loss of righting reflex, starting from

the time of pentobarbital injection is measured until the animal regains the righting reflex.

Mean value of duration of anesthesia in min are recorded in control and experiment group.

Fig 9: pentobarbital induced hypnotic test

Haloperidol induced catalepsy in mice[16]

Catalepsy in mice is induced by certain neuroleptic drugs due to their inhibitory effect on the

nigrostriatal dopamine system. It has been defined as a failure to correct an externally

imposed nasal posture over a prolonged period of time. After 30 minutes of drug

administration they are placed individually into a translucent plastic boxes with a wooden

dowel mounted horizontally 10 cm from the floor and 4cm from the end of the box, the floor

of the box is covered with approximately 2cm of the bedding material. The animals are

allowed to adapt to the box for 5min. thereafter each animal is grasped gently around the

shoulders and under the forepaws and placed carefully on the dowel. The amount of time

spent with at least one forepaw on the bar is determined. When the animal removes its paws,

the time is recorded and the mice is repositioned on the bar. Four trials are conducted for each

animal at 30, 60, 90, 120, an animal is considered cataleptic if it remains on the bar for 60


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sec. the percentage of cataleptic animals is calculated. The phenomenon of catalepsy predicts

antipsychotic activity.

Fig 10: haloperidol induced catalepsy

Step-down inhibitory avoidance[17]

Rodents when placed in an open field, are known to spend most of the time close to walls or

corners. When they are placed on an elevated platform in the center of rectangular

compartments, they instantaneously step down to the floor and explore the enclosure to

approach the wall or corner. The inhibitory avoidance model (IAM) was used to assess the

effects of the plant extract on the animals’ memory. In the training session (day 1),

immediately after stepping down and placing their paws on the grid, the animals received a

0.3 mA 2 s scrambled foot shock and were immediately withdrawn from the cage. Drugs

were given immediately after the training session to investigate their effect on memory

consolidation. Twenty-four hours later, in the test sessions (day 2), no foot shock was given

and the stepdown latency was used as a measure of retention (to a ceiling of 300 s).

Hole cross test[18]

The method was adopted as described by Takagi el al. (1971). A wooden partition was fixed

in the middle of a cage having a size of 30 x 20 x 14 cm. a hole or 3 cm diameter was made at

a height of 7.5 cm in the center of the cage. Diazepam (1mg/kg) is used as a standard drug.

After 30, 60, 90 min of administration of drugs, the mice were placed in the apparatus and the

number of passage of mouse through the hole from one chamber to other was counted for a

period of 3 min.


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Fig 11: hole cross test

Head dip test[19]

Boisser et al (1964) have used an open field with holes on the bottom into which the animals

cold poke their noses. The phenomenon indicates the curiosity component of the behavior of

mice. Swiss albino mice of either sex are used. The hole board has a size of 40 E and 40 cm

in which 16 holes of 3cm diameter each are evenly distributed on the floor. Nose pocking is

considered as an indication of curiosity/ exploratory behaviour and is measured by visual

observation or by electronic devices in more recent modifications. 30 minutes after

administration the animals are subjected to test for 5min. Diazepam is used as standard drug.

The average count of nose poking of treated animals is calculated as the percentage of control

animals.

Fig 12: head dip test

Isoniazid – induced convulsion[20]

Isoniazid is known to produce clonic – tonic seizures which can be used as one of the battery

of tests for anticonvulsant activity. Thirty minutes after i.p and 1 hour after oral

administration the animals are subjected to subcutaneous administration of isoniazid. During


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the next 2 hours the occurrence of colonic and tonic seizures and death is recorded. The

percentage of seizures and death occurring in control mice is taken as 100%. The suppression

of these effects in the treated group or the protection against death is calculated as percentage

of controls.

Fig 13: isoniazid induced convulsions

Effect of plant on normothermic animal[21]

Antidepressants have been shown to prevent the hypothermia induced by apomorphine in

mice. Compounds with a significant noradrenergic or dopaminergic activity are active against

apomorphine induced hypothermia but not the antidepressants which act mainly through

serotoninergic mechanism. A time vs temperature curve is constructed by mean temperature

of each group against time. The area under the curve (AUC) is calculated for all groups and

converted into percent inhibition of apomorphine induced hypothermia in.

Fig 14: effect of plant extract on normothermic animal control group.


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Antipsychotic induced weight gain [22]

The difference in the mice weight before the administration of drugs and after 0, 2, and 4

weeks of administrations of drugs was then compared.

Caffeine induced stereotypy and locomotion[23]

The method described by Randrup and Mnkvad (1967) was used for the stereotype

behavioural studies. Adult mice were randomized into five groups of 4 mice each. Group I

mice were received normal saline, group II mice were received caffeine as a control, group

III mice were given ethanolic bark extract of dolichonrone falcata (400mg/kg), group IV mice

were treated with ethanolic root extract of scrophularia hypericifolia (200mg/kg), group V

mice were treated with combination of both the extracts (DF+SH) and Group VI received

standard drug risperidone (1ml/kg) respectively. One hour after administration of saline and

extract and thirty minutes after administration of risperidone, group III, IV, V and VI mice

were given 2mg caffeine/kg. The signs of stereotype behaviour that included circling,

jumping, sniffing, and general locomotion were recorded for period of 2h using a hand held

tally counter (Irwin, 1968).

Stereotyped behaviour was scored as follows.

A. complete absence of stereotyped behaviour =0,

B. Presence of stereotyped movements of the head and intermittent sniffing = 1,

C. sniffing and chewing = 2,

D. chewing and intense licking = 3.

Evasion test [24]

The method of Turner (1965) was used, the animals were introduced into this rectangular box

with an inclined plane by which the mice can escape from the box, and the mice that escaped

within 5min from the rectangular box were selected for this test, 15 minutes after

administration of normal saline, diazepam as control, dolichondrone falcata and scrophularia

hypericifolia the animals (n=8) were placed in the box again and the number of mice

remaining in the box after 15 min in each group was noted.


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Fig 15: evasion test

RESULTS

phytochemical analysis of ethanolic bark extract of dolichondrone falcata

TABLE 1: Phytochemical analysis of ethanolic bark extract of dolichondrone falcata.

Types of constituents Ethanolic extracts

Alkaloids -

Flavonoids +

Steroids +

Phenolic compounds +

Carbohydrates +

phytochemical analysis of ethanolic root extract of scrophularia hypericifolia

TABLE 2: Phytochemical analysis of ethanolic root extract of scrophularia

hypericifolia.

Types of constituents Ethanolic extracts

Alkaloids +

Flavonoids +

Iridoid glycosides +

Phenylpropanoids +

Carbohydrates +

Steroid -


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Elevated plus maze.

TABLE 3: Effect of ethanolic extract of bark of dolichondrone falcata (test drug 1) and

root of scrophularia hypericifolia (test drug 2) and combination of both extracts (test

drug 3) and standard drug and standard drug on elevated plus maze test.

Group Drug treatment

Number of

entries in open

arms

Time spent in

open arm

Number of

entries in

closed arms

Time spent in

closed arms

Group I Normal saline (1ml) 2.750 ± 0.8539 1.500 ± 0.2887 3.500 ± 0.2887 1.000 ± 0.0

Group II Dolichondrone falcate

(DF)(400 mg/kg) 4.500 ± 0.5000 4.500 ± 0.2887 3.250 ± 0.4787 4.500 ±0.2887

Group III

Scrophularia

hypericifolia(SH)

(200mg/kg)

3.250 ± 0.2500 3.250 ± 0.2500 2.250 ± 0.2500 3.250 ± 0.4787

Group IV DF(200mg/kg) +

SH(100mg/kg) 3.500 ± 0.2887 3.500 ± 0.2887 2.250 ± 0.2500 3.500 ± 0.2887

Group V Diazepam(0.7mg/kg) 2.500 ± 0.2887 3.500 ± 0.2887 1.500 ± 0.2867 3.000 ± 0.4082

Data are presented as mean values (± S.E.M.). *P values: < 0.001 compared with treated

control. Statistical test employed was ANOVA.

Graph 1: open arm entries Graph 2: time spent in open arm

Graph 3: closed arm entries Graph 4: time spent in closed arm


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Locomotor activity

TABLE 4: Effect of ethanolic extract of bark of dolichondrone Falcata (test drug 1) and


drug 3) and standard drug on locomotor activity.

Group Drug treatment

Locomotor activity in 10 min Percentage

decrease in

activity Before

Treatment

After

Treatment

Group I Normal saline (1ml) 131.3 ± 5.154 138.3 ± 4.049 5%

Group II Caffeine (2mg/kg) 134.0 ± 3.719 138.8 ± 4.270 67.95%

Group III

Caffeine (2mg/kg) +

Dolichondrone falcata(DF)

(400mg/kg)

134.8 ± 2.287 58.75 ± 10.36 84%

Group IV

Caffeine (2mg/kg) +

Scrophularia

hypericifolia(SH) (200mg/kg)

1105 ± 7.089 46.50 ± 17.95 90.1%

Group V Caffeine (2mg/kg) + (DF

200mg/kg +SH 100mg/kg) 118.3 ± 3.065 22.75 ± 5.851 90.5%

Group VI Caffeine (2mg/kg) +

Diazepam(3mg/kg) 110.3 ± 3.705 24.25 ± 2.780 62%

(Observation period: 10 min) values are expressed as mean values ± S.E.M from 4 mice

significant at * *P < 0.01 as compared to control using one way ANOVA followed by

Dennett’s t- test.

Graph 5: locomotor activity score


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Novelty induced hypophagia



drug 3) and standard drug on novelty induced hypophagia.

Group Drug Dose/treatment Latency to drink (sec)

I Normal saline 1ml 321.8 ± 10.48

II Dolichondrone falcate(DF) 400mg/kg 207.3 ± 9.961

III Scrophularia hypericifolia(SH) 200mg/kg 243.8 ± 16.75

IV DF+SH 200mg/kg+ 100mg/kg 255.0 ±9.574

V Imipramine 10mg/kg 231.0 ± 5.260

Data are presented as mean values (± S.E.M.). *P < 0.05 compared with vehicle‐treated

control with ANOVA.

GRAPH 6: Novelty induced hypophagia test

Forced swim test



drug 3) and standard drug on forced swim test.

Group Drug Dose (mg/kg) Immobility (sec)

I Normal saline 1ml 137.5 ±1.190



IV DF + SH 200mg/kg + 100mg/kg 100.8 ± 4.732



control.


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GRAPH 7: forced swim test

Tail suspension test

TABLE 7: effect of ethanolic extract of bark of dolichondrone Falcata (test drug 1) and


drug 3) and standard drug on test.

Group Drug Dose Immobility(sec)




IV DF + SH 200mg/kg + 100mg/kg 97.50 ± 0.5000



control.

GRAPH 8: Tail suspension test


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Rota rod test



drug 3) and standard drug on Rota rod test.

Group Dose Time spend on rod in sec

0 sec 30 sec 60 sec 120 sec

I Normal saline (1ml) 262.5 ± 2.255 266.5 ± 5.606 262.5 ± 4.211 280.5 ± 6.357

II Dolichondrone falcate(DF)

(400mg/kg) 255.0 ± 2.887 137.5 ± 8.780 117.5 ± 11.09 130.0 ± 7.071

III Scrophularia hypericifolia(SH)

(200mg/kg 246.3 ± 8.985 125.0 ± 11.37 123.8 ± 25.44 132.5 ± 11.81

IV DF (200mg/kg)+SH

(100mg/kg) 259.0 ±5.115 128.8 ± 7.181 120.0 ± 4.564 123.8 ± 3.750

V Diazepam (5mg/kg) 263.8 ± 2.394 51.50 ± 2.901 43.50 ± 1.936 59.75 ± 4.090


control.

Graph 9: Rota rod test

Pentobarbital induced sleeping time



drug 3) and standard drug on pentobarbtone induced sleeping time.

Group Drug Dose/treatment mg/kg Onset of

sleep(min)

Duration of

sleep(min)

I Normal saline - - -

II Pentobarbital 30mg/kg 3.9 ± 0.33 26.9 ± 2.3

III Pentobarbital +Dolichondrone

falcate (DF) 30mg/kg + 400mg/kg 3.2 ± 0.24 41.1 ± 4.4

IV Pentobarbital + Scrophularia

hypericifolia (SH) 30mg/kg + 200mg/kg 5.2 ± 0.56 53.3 ± 6.5

V Pentobarbital + DF + SH 30mg/kg + 4.3 ± 0.15 80.3 ± 4.6


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200mg/kg+100mg/kg

VI Pentobarbital 30mg/kg +

Diazepam(5mg/kg) 30mg/kg + 4mg/kg 2.5 ± 0.16 100.7 ± 2.94


control.

Graph 10: Pentobarbital induced sleep time

Haloperidol induced catalepsy in mice

TABLE 10: Effect of ethanolic extract of bark of dolichondrone Falcata (test drug 1)

and root of scrophularia hypericifolia (test drug 2) and combination of both extracts

(test drug 3) and standard drug on haloperidol induced catalepsy.

Group Drug Mean cataleptic score(sec)

30 min 60 min 90 min 120 min

I NS (1ml) 25.50 ± 0.2887 51.50 ± 0.2887 115.0 ± 2.887 100.0 ± 0.0

II Haloperidol (1mg/kg) 26.25 ± 2.394 23.75 ± 9.437 33.75 ± 3.750 35.00 ± 4.564

III Haloperidol + Dolichondrone

falcate(DF) (400mg/kg) 5.500 ± 0.2887 3.500 ± 0.2887 3.500 ± 0.2887 7.250±0.4787

IV Haloperidol+Scrophularia

hypericifolia(SH) (200mg/kg) 11.00 ± 0.4082 5.000 ± 0.4082 14.75 ± 0.2500 13.50 ±0.866

V Haloperidol + DF (200mg/kg) +

SH (100mg/kg) 14.50 ± 0.2887 11.75 ± 0.2500 13.25 ± 0.4787 9.500±0.2887

VI Haloperidol + Risperidone

(1mg/kg) 6.750 ±0.2500 4.750 ± 0.4787 6.250 ± 0.2500 4.750±0.4787


control.


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Graph 11: Haloperidol induced catalepsy

Step down inhibitory avoidance



(test drug 3) and standard drug on step down inhibitory avoidance test.

Treatment Dose mg/kg Latency /time to step down in sec

I Normal saline(1ml) 193.8 ± 10.68

II Dolichondrone falcate (DF)400mg/kg 475.0 ± 20.62

III Scrophularia hypericifolia (SH)200mg/kg 473.8 ± 22.67

IV DF 200mg/kg+ SH 100mg/kg 307.5 ± 12.99

V Paracetamol (200mg/kg) 502.5 ±13.15


control.

Graph 12: step down test


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Head dip test



(test drug 3) and standard drug on head dip test.

Group Drug Dose No. of head

dipping


II Dolichondrone falcate (DF) 400mg/kg 22.25 ± 0.478

III Scrophularia hypericifolia

(SH) 200mg/kg 26.75 ± 0.2500

IV DF + SH 200mg/kg + 100 mg/kg 19.25 ± 0.4787

V Diazepam 2mg/kg 21.25 ± 0.4787


control.

Graph 13: head dip test

Hole cross test



(test drug 3) and standard drug on hole cross test.

Group Drug /Dose No. of movements (sec)

30 min 60 min 90 min

I Normal saline (1ml) 7.750 ± 0.2500 7.750 ± 0.2500 8.750 ± 0.2500

II Dolichondrone falcata (DF)

(400mg/kg) 1.500 ± 0.2887 2.000 ± 0.0 3.500 ± 0.5000

III Scrophularia hypericifolia

(SH) (200mg/kg) 4.000 ± 0.4082 3.000 ± 0.4082 4.000 ± 0.4082

IV DF (200mg/kg) + SH

(100mg/kg) 6.250 ± 0.4787 2.250 ± 0.4787 2.250 ± 0.4787

V Diazepam (1mg/kg) 1.250 ± 0.2500 6.250 ± 0.2500 4.250 ± 0.2500

Data are presented as mean values (± S.E.M.). *P < 0.001 compared with vehicle treated

control.


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Graph 14: hole cross test

Isoniazid induced convulsions



(test drug 3) and standard drug on isoniazid induced convulsions.

Group Drug Dose/treatment

mg/kg

Onset of

seizure

%

mortality

%

protection

I Normal saline - - - -

II Isoniazid 300mg/kg 81.2 ± 4.21 100 0

III Isoniazid+Dolichondrone

falcate (DF)

300 mg/kg+

400mg/kg 145.2 ± 6.2 0 100

IV Isoniazid +Scrophularia

hypericifolia (SH) 300mg/kg+200mg/kg 133.0 ± 6.38 25 75

V Isoniazid +DF + SH 300mg/kg+200mg/kg

+ 100 mg/kg 159.0 ± 6.38 0 100

VI Isoniazid+Diazepam 300mg/kg +2mg/kg A 0 100


control.

Graph 15: isoniazid induced convulsions


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Effect of drug on normothermic animals.

TABLE 15: Effect of ethanolic extract of bark of dolichondrone falcata (test drug 1)


(test drug 3) and standard drug on normothermic animals (mice).

Group Drug Dose/treatment

mg/kg

After drug administration

30min 60min

I Normal saline 1ml 36.55 ± 0.1100 36.66 ± 0.0

II Dolichondrone

falcate(DF) 400mg/kg 35.09 ± 0.5657 34.61 ± 0.1330

III Scrophularia

hypericifolia (SH) 200mg/kg 34.86 ± 0.1400 35.08 ± 0.05266

IV DF + SH 200mg+ 100mg 35.29 ± 0.1961 35.36 ± 0.1324


control.

Graph 16: effect of drug on normothermic animal

Antipsychotic induced weight gain.

Table 16: Effect of ethanolic extract of bark of dolichondrone Falcata (test drug 1) and


drug 3) and standard drug on antipsychotic induced weight gain in mice.

Group Drug / dose (mg/kg) Weight gain

0 weeks 2 weeks 4 weeks

I Normal saline(1ml) 23.75 ± 2.394 25.00 ± 0.0 24.75 ± 2.056

II Risperidone (1mg/kg) 24.00 ± 1.354 35.25 ± 2.056 39.50 ± 2.021

III Risperidone +dolichondrone

falcate (400mg/kg) 27.75 ± 1.315 37.00 ± 1.915 42.50 ± 1.443

IV Risperidone + scrophularia

hypericifolia (200mg/kg) 23.75 ± 1.250 36.75 ±1.974 45.50 ±1.658

V Risperidone + (DF200mg/kg

+ SH100mg/kg) 22.50 ± 1.443 36.75 ± 1.974 46.00 ± 1.354


control.


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Graph 17: weight gain

Caffeine induced stereotypy



(test drug 3) and standard drug on caffeine induced stereotypy.

Treatment Dose mg/kg Score for stereotypy

behavior

Control (caffeine) 2mg/kg 2.72 ± 0.09

Caffeine + Dolichondrone falcate 2mg/kg + 400 mg/kg 1.34 ± 0.05

Caffeine + scrophularia hypericifolia

(200mg/kg) 2mg/kg + 200mg/kg 1.22 ± 0.04

Caffeine + (DF 200mg/kg + SH

100mg/kg)

2mg/kg + 200mg/kg

+ 100mg/kg 0.66 ± 0.04

Caffeine + Haloperidol 2mg/kg + 1mg/kg 0.00 ± 0.0


control.

Graph 18: caffeine induced stereotypy


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Evasion test



(test drug 3) and standard drug on evasion test in mice.

Treatment Dose No. of mice remaining

in box after 5 min

Normal saline 1ml 0

Dolichondrone falcata (DF) 400mg/kg 3

Scrophularia hypericifolia (SH) 200mg/kg 2

DF + SH 200mg/kg +100mg/kg 6

Diazepam 2mg/kg 6


control.

Graph 19: evasion test

Histopathological Reports.

Fig 16: microphotograph of brain of mouse Fig 17: microphotograph of brain of mouse

treated with normal saline for one month with Ethanolic Bark Extract of

Dolichondrone falcata. one month.


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Fig 18: microphotograph of brain of

mouse treated with Ethanolic Root

Extract of scrophularia hypericifolia for

one month.

Fig 19: microphotograph of brain of

mouse treated with combination of

ethanolic bark extract of dolichondrone

falcata and root extract of scrophularia

hypericifolia.

Fig 20: microphotograph of brain of mouse treated with control group for one month.

DISCUSSION

The ethanolic extract of dolichondrone falcata and scrophularia hypericifolia gave positive

chemical reactions for glycosides, saponins, proteins, amino acids and flavonoids etc. No

toxic symptoms were observed for the drug up to of 400mg/kg body weight and 200mg/kg.

The extracts showed similar results when compared to standard drug diazepam. The

experimental result indicates that the extracts influence general behavior profile as evidenced

in awareness, alertness touch response, pinna reflex, righting reflex. However standard


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diazepam showed significant depression of all these responses. In Elevated plus maze in mice

Diazepam significantly increased the number of entries and time spent in open arms of

elevated plus maze. Dolichondrone falcata 400mg/kg and schrophularia hypericifolia

200mg/kg significantly increased the number of entries and percentage of time spent in open

arms as compared to vehicle (0.01% ethanol) treated mice. Diazepam and plant extracts

showed decrease in closed arm entries and time spent in closed arm. Therefore showed

significant antianxiety effect as compared to vehicle (0.01% ethanol) treated mice. However

scrophularia hypericifolia showed less result as compared to dolichondrone falcata.

Locomotor activity in mice showed that, both extracts significantly depressed the locomotor

activity at the tested dose level in mice. The activity was found to be maximum for

dolichondrone falcata and minimum for scrophularia hypericifolia. Further the activity of

extracts were more when compared with caffeine, a CNS depressant agent. In Novelty

induced hypophagia test on mice the latency to drink the condensed milk was decreased as

compared to normal saline. Both the extracts showed near similar results as that of standard

imipramine. In Forced swim test on mice drugs reliably decrease the duration of immobility

in animals during these tests. This decrease in duration of immobility is considered to have a

good predictive value in the evaluation of potential antidepressant agents (Porsolt RD,

et.al.1977). It was observed that the extract of Dolichondrone Falcata was more significant

than extract of Scrophularia Hypericifolia. In Tail suspension test on mice drugs reliably

decrease the duration of immobility in animals. This decrease in duration of immobility is

considered to have a good predictive value in the evaluation of potential antidepressant

agents (Porsolt RD, et.al.1977). It was observed that the extract of dolichondrone falcata was

more significant than extract of scrophularia hypericifolia.

The mean time duration on rotating rod was significantly decreased (P<0.001) and the

number of times animals falling down were increased in animals treated with Dolichondrone

falcata (400mg/kg) and Scrophularia hypericifolia (200mg/kg) as compared to control group,

indicating muscle relaxant activity and decline in motor coordination dolichondrone falcata

showed better result compared to scrophularia hypericifolia. However the extracts showed

good results in combination. In Pentobarbital induced sleeping test on mice, both extracts

increased sleep duration significantly modify the sleep latency. In agreement with the

previously published reports and as expected, diazepam significantly prolonged

pentobarbital-induced sleeping time, indicating that our study procedures were optimized.


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Both the extracts has protective effects against haloperidol induced catalepsy, which is

comparable to standard drug. In Step down inhibitory avoidance on mice the extracts showed

enhanced memory and learning behavior in mice which is showed by delay in the stepping

down of mice. Both the drugs increased the number of head pokes significantly (P < 0.01) as

compared to the reference standard (diazepam, 1 mg/kg, i.p.)-treated group showed

significant increase in exploratory activity. In the present study, the effect of ethanol extract

of dolichondrone falcata and scrophularia hypericifolia on CNS has been evaluated by hole

cross test. The result indicated that the extract significantly decreased locomotor activity

which indicates it has CNS depressant activity. Locomotor activity refers to an increase in

alertness and decrease in locomotor activity considered as sedative effect. The major

inhibitory neurotransmitter in the central nervous system is Gamma-amino-butyric acid

(GABA). Different types of anxiolytic, muscle relaxant, sedative-hypnotic drugs are shown

their action through GABAA, that’s why the extracts may acts by membrane

hyperpolarization which potentiating GABA-ergic inhibition in the CNS that leads to either

decrease in the firing rate of critical neurons in the brain or direct activation of GABA

receptor by the extracts

Most of conventional antiepileptic drugs are associated with many side effects such as

neurotoxic effects, cognitive deficits and teratogenic effects which decrease their clinical

utility (Trimble, 1987; Yerby, 1988; Meador et al., 1990). Recently, the search for novel

pharmacotherapy from medicinal plants for neurological and psychiatric diseases has

progressed significantly owing to their less side effects and better tolerability (Zhang, 2004.

Isoniazid exerts its convulsive effect by inhibiting GABA synthesis (Costa et al., 1975). It is

a potent monoamine oxidase (MAO) inhibitor and a glutamic acid decarboxylase (GAD)

inhibitor (enzyme involved in GABA synthesis) thus increases the brain monoamine content

and inhibited GABA synthesis respectively thereby leading to CNS excitation and

convulsions (Wood and Peesker, 1973; Marcus and Coulsto, 1985). Isoniazid induced

seizures was carried out to further confirm the GABA enhancing activity of the plant extract.

The extracts when compared to control treated produced significant increase in the time of

onset of clonic seizures, this shows dose-dependent increase in the anticonvulsant activity.

In present study the effect of the extracts on body temperature of mice were evaluated.

When compared with the normal saline the extracts showed a slight decrease in the

temperature, there was no change with the normal saline administration after 60 minutes.

http://scialert.net/fulltext/?doi=pjbs.2013.701.710&org=11#1108462_ja

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http://scialert.net/fulltext/?doi=pjbs.2013.701.710&org=11#37278_bc


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Effect of ethanolic bark extract of dolichondrone falcata and ethanolic root extract of

scrophularia hypericifolia on Caffeine induced stereotypic behavior on mice. In present

study, we investigated effects of extract of dolichondrone falcata and scrophularia

hypericifolia against caffeine induced stereotypy. Caffeine directly activates dopamine

receptors in the brain and doses of the drug induced stereotyped behaviour (sniffing, licking

and gnawing). The stimulant effect of high doses of APM is attributed to activation of post

synaptic receptors in the CNS. The behavioral responses observed in animals after

administration of the dopamine agonist, caffeine are attributed to activation of D1 and D2

receptors. Stereotyped behaviour is more closely associated with the caudate striatum area of

brain. Treatment of alcoholic extracts of bark and roots of dolichondrone falcata and

scrophularia hypericifolia extracts antagonized spontaneous motor activity and also caffeine

induced hyperactivity in mice. Findings indicate that the appetite stimulation–weight gain

associated with AAPDs is mediated by activation of hypothalamic AMPK linked to blockade

of the histamine H1R. AMPK stimulation parallels the orexigenic actions of the drugs, with

clozapine and olanzapine producing the most marked effects. The drug actions are very

potent, with substantial effects evident at 5 nm concentration. They are selective, with effects

restricted largely to the arcuate and paraventricular nuclei of the hypothalamus. Orexigenic

potencies of AAPDs parallel their affinities for histamine H1Rs, These findings are in accord

with studies implicating central histamine in weight control as well as the orexigenic role of

the paraventricular and arcuate nuclei. Moreover, mice, like humans, manifest weight gain in

response to AAPDs, although inhibition of locomotor activity sometimes impairs

characterization of orexigenic actions. The ethanolic extracts showed significant weight gain

in mice thus showing the property of antipsychotic drugs.

The extracts produced a significant decrease in exploratory behaviour pattern as evident from

the results of evasion tests. Positive control diazepam inhibited total residual curiosity. The

histopathological report shows the presence of cerebral edema, mild neuronal degeneration

when treated with the ethanolic plant extracts for one month, these abnormalities were due to

the induction of disease in mice. However with the control drug there was a glial reaction

which is generally considered to be consequence of neuronal death in neuro degenerative

disease such as Alzheimer’s, Huntington and Parkinson disease. The effect of plant ethanolic

extracts on mice brain showed absence of glial reaction which may be due to the inhibition of

glial reaction by plant extracts which thus represent a therapeutic target in treatment of many

psychological diseases.


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CONCLUSION

In conclusion, this study revealed that oral administration of ethanolic extract of bark of

dolichondrone falcata and roots of scrophularia hypericifolia exhibit psychoactive property.

However in most of the activities dolichondrone falcata showed higher results compared to

scrophularia hypericifolia. The drugs showed greater results when used in combination. The

plant material should be further investigated for use in humans to treat brain disorder.

ACKNOWLEDGEMENT

I’m thankful to Mrs. Mehnoor Farheen M.Pharm, Ph.D. project guide who has continuously

helped and gave valuable suggestion to guide me in successful completion of the project.

With great pleasure, I acknowledge my profound and sincere thanks to Principal Mrs.

Sunitha, Shadan women’s college of pharmacy, Hyderabad for inspiring me, I wish to

express my gratitude to beloved vice principal Mrs. Nishath Farheen M.Pharm, Ph.D. Shadan

women’s college of pharmacy, Hyderabad for constant encouragement throughout the period,

which helped me in the successful completion of the project.

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