PHYTOCHEMICAL EVALUATION AND PHARMACOLOGICAL … · PHYTOCHEMICAL EVALUATION AND PHARMACOLOGICAL SCREENING OF ETHANOLIC LEAF EXTRACTS OF EICHHORNIA CRASSIPES AND NELUMBO NUCIFERA

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874

Farheen et al. World Journal of Pharmacy and Pharmaceutical Sciences

PHYTOCHEMICAL EVALUATION AND PHARMACOLOGICAL

SCREENING OF ETHANOLIC LEAF EXTRACTS OF EICHHORNIA

CRASSIPES AND NELUMBO NUCIFERA FOR

NEUROPHARMACOLOGICAL ACTIVITY IN PSYCHONEUROSIS

INDUCED MICE

Mehnoor Farheen*1, Syeda Arman R. O. Hussaini

2, Asfia kouser

3

1*

Department of Pharmacology, Assistant Professor Shadan womens College of Pharmacy,

JNTU, Hyderabad.

2,3M. Pharm [Pharmacology], Shadan Womens College of Pharmacy, JNTU, Hyderabad.

ABSTRACT

Behavioral neuropharmacology focuses on the study of how drugs

affect behavior in humans and animals. This study assessed the

sedative, CNS depressant, Analgesic, Anti-epileptic, Anti-anxiety,

Anti-psychosis, Anti depressant and Memory enhancing properties of

ethanolic extract of Nelumbo nucifera, Eichhornia crassipes and

combination of ethanolic extracts of Nelumbo nucifera and Eichhornia

crassipes in disease induced mice. Extraction of ethanolic extract from

leaves of Nelumbo nucifera and Eichhornia crassipes 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 consisted of mice induced with

depression, anxiety, psychosis, epilepsy and were treated with Normal

saline. Group III, IV and V consisted of normal mice induced with depression, anxiety,

psychosis, epilepsy. Oral administration of extract resulted in significant sedative, CNS

depressant, Analgesic, Anti-epileptic, Anti-anxiety, Anti-psychosis, Anti depressant and

Memory enhancing effects. Group VI consisted of normal mice induced with depression,

anxiety, psychosis, epilepsy and were treated with standard drug. The effects produced by the

extracts were closely similar to the standard drugs. In conclusion, the present study indicates

that the ethanolic leaf extracts of Nelumbo nucifera and Eichhornia crassipes and

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

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Volume 4, Issue 12, 874-904 RReesseeaarrcchh Article ISSN 2278 – 4357

Article Received on

05 Oct 2015,

Revised on 29 Oct 2015,

Accepted on 22 Nov 2015

*Correspondence for

Author

Mehnoor Farheen

Department of

Pharmacology, Assistant

Professor Shadan womens

College of Pharmacy,

JNTU, Hyderabad.


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combination of ethanolic extracts of Nelumbo nucifera and Eichhornia crassipes appears to

exhibit sedative, CNS depressant, Analgesic, Anti-epileptic, Anti-anxiety, Anti-psychosis,

Anti depressant and Memory enhancing activity in disease induced mice.

KEYWORDS: Anti-psychosis, CNS Depressant, Depression, Eichhornia crassipes, Memory

enhancing, Neuropharmacology.

1. INTRODUCTION

Neurological disorders cause many physical, mental, emotional and social stresses to

individuals suffering from them. Indivituals suffering from a neurological disorder or

disorders may experience many serious, long-term mental complications. These

complications give rise to social inadequacies which can gradually worsen with time. Mental

health is an important issue which is not given enough attention, thereby increasing the

number of mental health patients by the day. Data has shown a disturbing increase in

numbers of mental health issues in just 5 years (2005 to 2010).[1]

Mental disorders are the

core health challenge of the 21st century. Most Neurological disorders are preventable and

easily treatable with drugs. These drugs come with a number of complications associated

with them which can worsen the patients mental health and be very pricey, causing patients to

avoid over-all therapy. Patients search for natural substances which can give relief as well as

be cost-effective, this is where researches such as this one, come into play. Medicinal plants

have attracted global interest in recent years. In the past few decades, pioneer work in

identification, documentation and recognition of traditional medicine has been done in India.

Investigation of traditional medicine is very important for the welfare of rural and tribal

communities for the treatment of conventional illness. In this study, Nelumbo nucifera,

Eichhornia crassipes and combination drug consisting of both Nelumbo nucifera and

Eichhornia crassipes was evaluated for its use as a sedative, CNS depressant, Analgesic,

Anti-epileptic, Anti-anxiety, Anti-psychosis, Anti depressant and Memory enhancing

substance.

Review of Drug under study

Nelumbo nucifera

Nelumbo nucifera, also known as Indian lotus is one of two species of aquatic plant in the

family Nelumbonaceae. In Ayurveda Nelumbo nucifera is used as a diuretic and anthelmintic

and in the treatment of strangury, vomiting, leprosy, skin diseases and nervous

exhaustion.[2,3,4]

In popular medicine it is used in the treatment of tissue inflammation, cancer,


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skin diseases, leprosyand as a poison antidote.[5,6]

Rhizomes are prescribed asdemulcents for

haemorrhoids and are beneficial in dysentery,chronic dyspepsia, and have nutritive, diuretic

and cholagogueactivities.[7,8]

The leaves are usedfor the treatment of haematemesis, epistaxis,

haemoptysis,haematuria, metrorrhagia and hyperlipidaemia.[9]

The flowersare useful in the

treatment of diarrhoea, cholera, fever and gastriculcers[5]

The seeds and fruits are used as a

health food in Asia and to treat many ailments, including poor digestion, enteritis, chronic

diarrhea, insomnia, palpitations, spermatorrhoea, leucorrhoea, dermatopathy, halitosis,

menorrhagia, leprosy, tissue inflammation, cancer, fever and heart complaints, and as

anantiemetic, poisoning antidote, diuretic and refrigerant.[5,10,11,12]

Lotus seedpods are

sometimes used as a traditional medicine for haemostatic function.[13]

The seed powder

mixed with honey is useful in treating cough.[3]

Embryos of lotus seed are used in traditional

Chinese medicine to overcome nervous disorders, insomnia, high fevers (withrestlessness)

and cardiovascular diseases (e.g. hypertension, arrhythmia).[14]

Eichhornia crassipes

Eichhornia crassipes, commonly known as (common) water hyacinth, is an aquatic plant

native to the Amazon basin, and is often considered a highly problematic invasive species

outside its native range. This invasive nuisance is planta non grata in much of the world

where it often jams rivers and lakes with uncounted thousands of tons of floating plant

matter. A healthy acre of water hyacinths can weigh up to 200 tons. the water hyacinth in

most places is under "maintenance control": field crews constantly working to keep the plant

numbers at their lowest possible levels, in exchange for the rivers and lakes remaining usable.

Eichhornia crassipes is used for Bioenergy, phytoremediation, food and as animal feed and

fertilizer. Studies show, it can be used as Anti-algal [15]

, Anti-Microbial [15]

and

Antioxidant.[16]

This research has tested the efectivity of the two plants, Nelumbo nucifera

and Eichhornia crassipes, for their neuropharmacological property. The findings, were

impressive.

MATERIALS AND METHODS

Collection of drugs

Dried leaves of both plants Eichhornia crassipes (Pontederiaceae) and Nelumbo nucifera

(Nelumbonaceae) were collected. The plants were taxonomically identified and authenticated

by DR. K. MADHAVA CHETTY Assistant Professor of Botany, Department of

Pharmacognosy, Sri Venkateshwara University, Tirupathi.


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Preperation of plant extract

The leaves were cut into pieces and subjected to shade drying. On complete drying, the

pieces were powdered and stored in air tight containers at room temperatures. The powdered

was macerated with ethanol for 7 days and then filtered. The filtrate was evaporated to obtain

dried extract. The extract thus obtained was subjected for evaluation of neuropharmacological

and analgesic activity study. The plant extract was prepared by maceration process.

Maceration: In maceration(for fluid extact), whole or coarsely powdered plant-drug is kept in

contact with the solvent in a stoppered container for a defined period with frequent agitation

until soluble matter is dissolved. This method is best suitable for use in case of the

thermolabile drugs. Using this process, 500g/kg powder was added in ethanol in the ratio 1:2

and vigorous shaking was carried out for 7 days continuously and was kept at room

temperature. The filtrate thus obtained was ethanolic extract. The filtrate obtained was

dissolved in 0.9% normal saline and used as vehicle in the experiment.

Preliminary phytochemical screening

Standard screening test of the extract was carried out for various plant constituents. The crude

extract was screened for the presence or absence of secondary metabolites such as in

Alkaloids, flavonoids, sterols,Glycosides, terpenoids, anthroquinone, proteins, phenols and

anthocyanins using standard procedures.

Acute toxicity studies

Acute toxicity study was performed for the extract according to the acute toxic classic

method as per the method of Litchfield & Wilcoxon (1949). Acute toxicity study was carried

out on plant extracts using Male and Female Albino mice. The mice were fasted overnight

and the weight of each mouse was recorded just before use. The animals were divided into

five groups containing 6 animals each, the extract was administered orally in increasing dose

up to 2000 mg/kg b.w. After the treatment the animals were observed for mortality or toxicity

for 72 hours. No changes in skin and fur, eyes, autonomic (salivation, lacrimation,

defecation) and central nervous system (ptosis, drowsiness, gait, tremors and convulsions)

were observed. No deaths were observed.

Experimental animals

Albino mice (15-50 gms) of either sex housed in standard conditions of temperature (55 ±

5%) and light (12 hrs light/dark cycles) were used. They were fed with standard pellet diet


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and water and libitum. Animals were randomly selected for grouping. All experiments were

performed according to the norms of the ethical committee (CPCSEA).

SCREENING METHODS

Head dip test

A specially designed rectangular-shaped head dip box having 15 holes in each side was used

in this study. The number of head dips by mice through these holes in a specified time was

counted. Mice of groups II, III, IV received Test drug 1, test drug 2 and combination of test

drugs 1 and 2 respectively. The groups I and V received control and standard drug Diazepam

(2 mg/kg, i.p). The control and drug-treated animals were placed individually in the head dip

box and the observations were made for 30 min[17][18][19]

Locomotor Activity

It can be studied with the help of an Actophotometer. Each mouse was placed individually in

actophotometer & the basal activity score of the animals were recorded after 30, 60 & 120

mins of drug treatment. Groups III, IV, V received Test drug 1, test drug 2 and combination

of test drugs 1 and 2 respectively. The groups I, II and V received control, Normal saline and

standard drug Diazepam (3 mg/kg, i.p ). Activity on each mouse was retested for 10mins.

Difference in the activity was recorded considering before treatment values & after treatment

values. Finally, Percentage decrease in locomotor activity was Calculated[20]

Rota-rod test (Thread-mill device test)

The study was carried out according to the method described by Perez et al., (1998). Rota rod

treadmill device was used for this experiment. Mice trained to remain on slowly moving (16

rpm) rods of 5 cm diameter for 180 seconds or longer were selected. . Groups II, III, IV

received Test drug 1, test drug 2 and combination of test drugs 1 and 2 respectively. The

groups I and V received control and standard drug Diazepam (10 mg/kg, i.p ). One hour after

administration of test drugs, the mice were placed singly on the rod for 3 minutes, at 30

minutes intervals for 3 h. If an animal failed more than once to remain on the rod for 3

minutes, the test was considered positive, meaning that there is lack of motor coordinations.

Pentobarbitone induced sleep test in mice

The method of Rakotonirina et al. (2001), was adopted. Sleep potentiation time of the test

drugs was studied in group of mice that received Pentobarbitone at a dose of 30 mg kg body

weight one hour after i.p. administration of Teat drug 1(Group II), test drug 2(Group III),


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combination of both test drugs(Group IV), standard drugs Diazepam(Group V) and Normal

saline(Group I); with 4 mice in each group. The sleeping time was estimated as the time

between disappearance and recovery of the straightening reflex.

Hole cross test

The method was adopted as described by Takagi et al. (1971). A partition was fixed in the

middle of a cage having a size of 30 × 20 × 14 cm. A hole of 3 cm diameter was made at a

height of 7.5 cm in the centre of the cage. The number of passage of a mouse through the

hole from one chamber to the other was counted for a period of 3 min at 0, 30, 60, 90 and 120

min after oral administration of Normal saline(Group I), the test drugs(Group II, III), their

combination(Group IV) and standard drug( Diazepam 1mg/kg b.w)(Group V). (Takagi et al.,

1971).

Hot plate method

The method was adopted as described by Sachin Kumar Jain et al., 2014. The mice of either

sex were weighed and divided into five groups (n = 4 in each groups). Group I served as

control. Group V (Aspirin 10 mg / kg body weight) served as standard and group II, III and

IV were treated with test drug 1, test drug 2 and combination of test drugs 1 and 2

respectively. Reaction time of animals was noted down in hot plate at 0, 30, 60, 90 and 150

minutes after the treatment. The basal reaction time taken by observing hind paw licking or

jump response (whichever appear first) in animals while placed on hot plate,which was

maintained at constant temperature 55ºC ± 1ºC. A cut off period of 15 seconds was observed

to avoid damage to the paws. The percentage of analgesia was calculated.

Acetic acid-induced writhing in mice

The experiment was carried out according to the method of Witkin et al. Test drugs were

administered by the oral route one hour prior to the injection of acetic acid. Writhing was

induced in animals by injecting acetic acid 300 mg/kg (3% solution in sterile distilled water)

i.p. Each mouse was then kept indivitually and the total number of writhing episodes for a

period of 20 minutes after the injection of acetic acid was counted. The percent inhibition of

the writhing count of the treated group was calculated from the mean writhing count of the

control group.


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Tail flick test

Albino mice were fasted for 24 hrs with water maintained at room temperature. Mice were

treated by oral gavage with Normal saline(Group I), Test drug 1(Group II), test drug 2(Group

III), combination of test drugs 1 and 2(Group IV) and standard drug(Group V), Aspirin (100

mg/kg, i.p). Nearly 1 or 2 cm of the tail was immersed in warm water kept constant at 50 ºC.

The reaction time was the time taken by the mice to deflect their tails. The first reading was

discarded and the reaction time was taken as the mean of the next two readings. The latent

period of tail flick response was taken as the index of anti-nociception and was determined at

15, 30, 60 and 90 min after the administration of drugs. The maximum reaction time was

fixed at 15 sec (Sewel and Spencer., 1976).

Isoniazid Induced Convulsions

Isoniazid at the dose of 300 mg/kg was injected i.p. to induce clonic-tonic convulsions in

mice. Groups II, III, IV received Test drug 1, test drug 2 and combination of test drugs 1 and

2 respectively. The groups I and V received control, and standard drug Phenytoin (10 mg/kg,

i.p ). Each animal was placed in individual plastic cage for observation lasting forty five

minutes. The parameters noted were mean onset time of convulsions, duration of convulsions

and number of deaths (% mortality).

Elevated plus Maze test

Anxiety-related behavior was measured by the elevated plus-maze test. The elevated plus-

maze Consist of two open arms, 50 × 10 cm, and two closed arms, 50 × 10 × 40 cm. The

maze was elevated to a height of 15 cm above the floor. Groups II, III, IV, received Test drug

1, test drug 2 and combination of test drugs 1 and 2 respectively. The groups I and V received

control, Normal saline and standard drug Diazepam (35 mg/kg, i.p ). Each mouse was placed

on the central platform facing a closed arm. During 5 min test period, the following measures

were taken by an observer, number of open arms entries, time spent in open arms, number of

closed arm entries, time spent in closed arms. Entering into an arm was noted only when all

paws had crossed out central area. (Kurt, M.,2004).

Novelty Induced Hypophagia

Albino Mice were divided in to five groups (n=6). Three days was allowed for habituation by

exposing mice to sweetened milk (Carnation sweetened condensed milk, 1:3 in water) in the

home cage (~1-2 hr sessions). On the fourth day, latency to drink from the sweetened milk

bottles in their home cage (dim lighting) was noted. On the fifth day drug administration of


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Test drug 1, test drug 2 and combination of test drugs 1 and 2 and standard drug to mice of

groups II, III, IV and V respectively were done followed by measuring latencies to drink the

sweetened milk in a novel cage (bright lighting). Comparisons of drinking latencies between

home cage and novel cage were made.(Merali et al., 2004).

Evasion test

The method of Turner (1965) was used. The animals were introduced into a rectangularbox

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(Group I), diazepam(0.5mg/kg) (Group V) as standard and

Test drugs1(Group II), 2(Group III) and combination of both(Group IV), the animals were

placed in the box again and the number of mice remaining in the box after 15 min in each

group was noted.

Caffeine-induced stereotypy and locomotion

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

behavioural studies. Adult mice were randomized into five groups of six mice each. Groups

III, IV, V received Test drug 1, test drug 2 and combination of test drugs 1 and 2 respectively.

The groups I, II and V received control, Normal saline and standard drug chlorpromazine (2

mg/kg, i.p ).. One hour after administration of saline and extract and thirty minutes after

chlorpromazine administration, all the mice were given 2 mg caffeine/kg. The signs of

stereotype behaviour that included circling, jumping, sniffing, and general locomotion were

recorded for a period of 2 h using a hand held tally counter (Irwin, 1968).

Stereotyped behavior was scored as follows.

-Complete absence of stereotyped behavior, presence of stereotyped movements of the

head and intermittent sniffing= 1,

-Sniffing and chewing= 2;

-Chewing and intense licking= 3.

Locomotion was assessed using the method of Sturgeon et al. (1979) simultaneously with the

stereotypy and scored as follows.

-Stationary= 0;

-Movements within a localized area, forelimbs only= 1;

-Intermittent movements within half of the area of the cage= 2;


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-Continuous movements within half of the area of the cage= 3;

-Intermittent movements within the whole area of the cage= 4;

-Continuous movements within the whole area of the cage= 5.

Catalepsy in mice

Five groups of 4 albino mice each weighing between 20-50g are used. They are treated with

the test drug or the standard given intraperitoneally i.e. Groups II, III and IV received Test

drug 1, test drug 2 and combination of test drugs 1 and 2 respectively. The groups I and V

received Normal saline and standard drug Resperidone (1 ml/kg, i.p ). After 30 min they are

placed individually into translucent plastic boxes with a dowel mounted horizontally 15cm

from the floor and 5cm from the end of the box. The floor of the box is covered with

approximate 8cm of the bedding material. The animals are allowed to adapt to the box for 5

min. Thereafter each animal is grasped gently around the shoulders and the forepaws are

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 paw, the time is recorded and the mouse is

repositioned on the bar. Four trials are conducted for each animal at 30, 60, 90, 120mins. An

animal is considered cataleptic if it remains on the bar for 60 seconds or more. The

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

antipsychotic activity as well as the potential of producing extrapyramidial side effects[21]

Antipsychotic induced weight gain

Mice were randomly divided into five groups. Each mouse was individually weighed before

administration of drugs. Hereafter, mice of groups I, II, III, IV and V received Normal saline,

Resperidone (control), Test drug 1, test drug 2 and combination of test drugs 1 and 2

respectively everyday for 4 weeks. Normal mice chow feed and water were provided as

usual. After 2 and 4 weeks, mice were individually weighed again. The difference in the mice

weight before the administrations of drugs, after 2 weeks and after 4 weeks of administrations

of drugs was then compared.

Forced Swim Test (FST)

In this test mice are forced to swim in a restricted space from which there is no escape and

become immobile. Individual mice were forced to swim in an open cylindrical container

containing 7 cm of water at 22.0 ±1.0ºC; the duration of immobility or struggling in a period

of 6 minutes was recorded. Immobility was evaluated as when mice ceased to struggle and

remained floating in the water, making only necessary movements necessary to keep its head


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above water. At the end of the session, the animal was removed from water and dried

gently.[23]

Mice of Group I, II, III, IV, V received Normal saline, Test drug 1, Test drug 2,

combination of test drugs 1 and 2 and standard drug (Imipramine 10mg/kg) respectively.

Tail suspension test

Mice of Group I, II, III, IV, V received Normal saline, Test drug 1, Test drug 2, combination

of test drugs 1 and 2 and standard drug (Imipramine 10mg/kg) respectively. The total

duration of immobility induced by tail suspension was measured according to the method

described by (Steru et al.). Mice were suspended on the edge of a table 50 cm above the floor

by the adhesive tape placed approximately 1 cm from the tip of the tail. The total duration of

immobility was recorded for a total 6 min.

Step down method

Mice of either sex were used for this test. A rectangular box 50×50×50cm with grid floor and

a movable platform in the center of the box is used. The grid floor is connected to a shock

device, which delivers scrambled foot shocks. Mice of groups II, III and IV received Test

drug 1, test drug 2 and combination of test drugs 1 and 2 respectively. Groups I and V

received Normal saline and standard drug(Piracetam 100mg/kg).

The actual experiment comprises of three stages. In the first stage of familiarization, the

animal is placed on the platform, which is released after raising the cylinder and a latency to

descend is measured. After 10 sec of exploration, it is returned to the home cage. During the

second stage of learning a foot shock (50Hz; 1.5mA; 1s) is administered as soon as the

animal descends from the platform and the animal is then returned to the home cage. In the

final stage of retention test, the animal is again placed on the platform after 24 hours of the

learning trial and the latency to step down is measured.

The test is concluded when the animal steps down or remains on the platform for a cut-off

time of 60 seconds. The average time of descent during the learning stage and during the

reaction test are measured.

Prolongation of the step-down learning is defined as latency. However, the variability of

results obtained by this method is very high and it is necessary to use a large number of

animals. The animals must be handled very gently and the foot-shock must be delivered


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immediately at the first contact of the animal with the floor. Further the room must be kept

sound proof during the entire procedure.[22]

Histopathological reports

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

isolated brains were stored in labeled containers containing neutral formalin (10% solution).

These brains were submitted to VIJAYA DIAGNOSTIC CENTER for further

histopathological studies.

RESULTS AND DISCUSSION

Table 1: Phytochemical Analysis of Ethanolic leaf extract of Nelumbo nucifera and

Eichhornia crassipes.

Constituent Nelumbo

nucifera

Eichhornia

crassipes

Alkaloids + +

Flavonoids + +

Steroids + +

Glycosides + +

Proteins + +

Phenolic compounds + +

Saponins - -

Triterpenoids - +

Carbohydrates + -

Tannins + -

(+) indicates presence; (-) indicates absence

Table 2: Effect Of Ethanolic Extract Of Leaves Of Nelumbo Nucifera (Test Drug 1),

Eichhornia Crassipes (Test Drug 2), Combination Of Both Test Drugs (Test Drug 3)

And Standard Drug On Head Dip Test.

S.no Treatment Number of head dips

1 Normal saline 39.25±0.4787

2 Nelumbo nucifera 13.50±0.9574

3 Eichhornia crassipes 11.25±0.25

4 Nelumbo nucifera +

Eichhornia crassipes 8.5±0.2887

5 Standard

(Diazepam 2mg/kg) 13.75±0.8539


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The exploratory activity decreases significantly when doses of ethanolic leaf extract of

Nelumbo nucifera, Eichhornia crassipes and combination of both Nelumbo nucifera and

Eichhornia crassipes are given. The decrease effect is higher than that of mice treated with

Diazepam as standard drug.

Table 3: Effect of Ethanolic Extract Of Leaves Of Nelumbo Nucifera (Test Drug 1),


And Standard Drug On Locomotor Activity.

S.no Treatment Locomotor activity in 10 mins

Before treatment After treatment Decrease %

1 Vehicle 96±21.33 96.75±20.66 -0.78%

2 Control

(Caffeine 40mg/kg) 91.25±4.049 160.3±7.016 -75.61%

3 Nelumbo nucifera 97.75±1.601 59.25±0.4787 39.38%

4 Eichhornia crassipes 132.8±6.074 69.25±0.4787 47.83%

5

Nelumbo nucifera +

Eichhornia crassipes 43±1.780 39.75±0.8539 7.55%

6 Standard

(Diazepam 3mg/kg) 101.5±1.190 85±1.080 16.25%


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Percentage decrease in locomotor activity was highest in mice treated with ethanolic extract

of leaves of Eichhornia crassipes. Ethanolic extracts of Nelumbo nucifera and and

combination of both Nelumbo nucifera and Eichhornia crassipes also showed high decrease

percentage in locomotor activity when compared to control and Standard drug Diazepam.



And Standard Drug On Rota-Rod Test

S.no Treatment Duration of stay on rota-rod

0 mins 30 mins 60 mins 120 mins

1 Vehicle 262.5±2.255 266.5±5.60 262.8±4.211 268±6.519

2 Nelumbo nucifera 267.3±6.725 312.8±12.10 226.8±13.58 260±5.788

3 Eichhornia crassipes 260±8.436 66±7.714 48.75±0.6296 193.8±2.136

4

Nelumbo nucifera +

Eichhornia crassipes 266±5.115 200.8±1.548 226.3±2.213 274±9.065

5 Standard

(Diazepam 10mg/kg) 267.5±3.862 55.75±1.493 44.50±1.555 105±2.483

The extract-treated mice were able to maintain their posture on the rotating rod without

falling for over 180 seconds, especially of those mice treated with Combination of both tests

drugs and that of Nelumbo nucifera and the cut-off time on the tread mill was used.


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Table 5: Effect of Ethanolic Extract of Leaves Of Nelumbo Nucifera (Test Drug 1),


And Standard Drug On Pentobarbitone Induced Sleep Test.

S.no Treatment Sleep time (Mins)

Onset of sleep Duration of sleep Prolongation %

1 Control

(Pentobarbitone) 21.25±1.25 44.25±1.548 ---

2 Nelumbo nucifera 17.5±1.443 52.75±5.662 19.20%

3 Eichhornia crassipes 8.25±0.6292 70.5±0.6455 59.32%

4

Nelumbo

nucifera+Eichhornia

crassipes

9.5±0.5 54.5±1.848 23.16%

5 Standard

(Diazepam 3mg/kg) 2.25±0.6292 110.3±4.090 149.15%

The ethanolic extract of Eichhornia crassipes and combination of bith test drugs significantly

shortened the sleep onset time (sleep latency) and prolonged sleeping time. They both also

significantly prolonged duration of sleep.

Table 6: Effect of Ethanolic Extract of Leaves of Nelumbo Nucifera (Test Drug 1),

Eichhornia Crassipes (Test Drug 2), Combination of Both Test Drugs (Test Drug 3) And

Standard Drug On Hole Cross Test.

S.no Treatment Number of movements (= number of hole cross)

0 min 30 min 60 min 90 min

1 Vehicle 6.75±0.75 7.5±0.2887 9±0.4782 7.5±0.2887

2 Nelumbo nucifera 5.5±0.2887 0.5±0.2887 3.5±0.2887 2.5±0.2887

3 Eichhornia crassipes 5±0.4082 1±0.0 5±0.4082 3.75±0.25

4

Nelumbo nucifera +

Eichhornia crassipes 5.75±0.8539 1.5±0.2887 5.5±0.2887 5.5±0.50

5

Standard

(Diazepam 1mg/kg) 4.75±0.4787 1.25±0.4787 1.5±0.6455 0.5±0.2887


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The ethanolic extracts of all three test drugs (Nelumbo nucifera, Eichhornia crassipes and

combination of Nelumbo nucifera and Eichhornia crassipes) significantly displayed a

suppression of motor activity and exploratory behavior in this test. The locomotor activity

lowering effect was evident in the second (30 min) and third observation (60 min) and

continued up to a fifth observation period (120 min).



And Standard Drug On Hot Plate Test.

S.no Treatment Reaction time in seconds

0 min 30 mins 60 mins 90 mins 150 mins

1 Vehicle 0.5±0.2887 1±0.0 0.5±0.2887 0.5±0.2887 1±0.0

2 Nelumbo nucifera 7±0.4082 7±0.8165 7±1.225 6.25±0.25 10±0.4082

3 Eichhornia crassipes 4±0.4082 7.5±0.2887 7.75±0.8539 5±1.080 8±0.8165


Eichhornia crassipes 7.75±0.4787 3.7±0.4787 16±0.7071 8±0.0 7.25±0.25

5

Standard

(Aspirin 10mg/kg) 5±0.4082 14.25±0.8539 14.25±0.8539 14.25±0.4787 15.75±1.493

All three doses of the ethanolic extracts of test drugs produced increase in latency time

when compared with the vehicle. At the third observation (60 mins), ethanolic extract of


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combination of Nelumbo nucifera and Eichhornia crassipes produced higher latency period

than the standard drug Aspirin.



And Standard Drug On Acetic Acid Induced Writhing Test.

S.no Treatment No. of writhings Inhibition %

1 Control

(Acetic acid 300mg/kg) 31.50±1.323 ---

2 Nelumbo nucifera 14.25±0.8539 54.76%

3 Eichhornia crassipes 16.25±2.056 48.41%


Eichhornia crassipes 6.25±0.8539 80.15%

5 Standard

(Ibuprofen 40mg/kg) 3.75±0.8539 88.09%

The administration of all three test drug extracts significantly inhibited writhing response

induced by acetic acid when compared to mice administered with Vehicle. Inhibition

percentage was found to be highest in combination of ethanolic extracts of Nelumbo nucifera

and Eichhornia crassipes.


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And Standard Drug On Tail Flick Test.

S.no Treatment Tail withdrawal time in seconds (Mean ± SEM)


1 Vehicle 0±0.0 0.25±0.25 1±0.4082 0.75±0.4787

2 Nelumbo nucifera 4.5±1.041 4.75±1.25 3.5±1.041 3.25±0.75

3 Eichhornia crassipes 3.25±0.8539 3.75±0.8539 2.75±0.4787 1.75±0.25


Eichhornia crassipes 4.75±0.8539 8.5±1.323 6.5±0.866 4.75±0.75

5 Standard

(Aspirin 100mg/kg) 3.75±0.4787 2.5±0.6455 2.5±0.2887 2±0.0

The tail withdrawal reflex time following administration of the ethanolic extracts of test

drugs was found to increase. The tail withdrawal time of mice administered Nelumbo

nucifera and combination of both drugs i.e, Nelumbo nucifera and Eichhornia crassipes was

observed to be higher than that of mice administered standard drug, Aspirin at all 4

observations (15 mins, 30 mins, 60 mins, 90 mins)

Table 10: effect of ethanolic extract of leaves of nelumbo nucifera (test drug 1),

eichhornia crassipes (test drug 2), combination of both test drugs (test drug 3) and

standard drug on isoniazid induced convulsion test.

S.no Treatment Onset of

seizures

Duration of

seizures

Number of

deaths Mortality %

1 Control

(Isoniazid 300mg/kg) 612±4.416 39.25±0.4787 4/4 100%

2 Nelumbo nucifera 651.3±1.315 35.75±2.287 2/4 50%

3 Eichhornia crassipes 697.8±1.702 33.25±1.797 1/4 25%

4 Nelumbo nucifera + 781.5±1.19 24.25±0.8539 0/4 0%


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5 Standard

(Phenytoin 10mg/kg) 874±2.677 7.25±1.031 0/4 0%

Time of onset of seizures of all three drug administrated mice was observed to have increased

when compared to control. Duration of seizures steadily decreased when compared to control

as well. Highest increase in time ofonset of seizures was found in mice treated with

combination of ethanolic extracts Nelumbo nucifera and Eichhornia crassipes.



And Standard Drug On Elevated Plus Maze Test.

S.no Treatment

Number of entries in

open arms (Seconds)

(Mean ± SEM)

Time spent in open

arms (Seconds)

(Mean ± SEM)

1 Vehicle 1±0.0 36.25±8.004

2 Nelumbo nucifera 3.75±0.4787 126.8±2.287

3 Eichhornia crassipes 4.25±0.25 199.3±13.73


Eichhornia crassipes 4.5±1.5 215±31.32

5 Standard

(Diazepam 35mg/kg) 3.75±0.4787 234.3±5.105

Table 11 a:

S.no Treatment

Number of entries in

closed arms (Seconds)

(Mean ± SEM)

Time spent in closed

arms (Seconds)

(Mean ± SEM)

1 Vehicle 2.75±0.25 263.8±8.004

2 Nelumbo nucifera 4.75±0.4787 173.3±2.287

3 Eichhornia crassipes 3.75±0.9465 100.8±13.73

4 Nelumbo nucifera + 1.5±0.5 83.75±31.32


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5 Standard

(Diazepam 35mg/kg) 1.25±0.25 65.75±5.105

The ethanolic extracts of Nelumbo nucifera and Eichhornia crassipes and combination of

both test drugs significantly increased the duration of stay of mice in open arm of apparatus

and decreased the duration of stay in the closed arm. Most time spent in open arm was

observed in mice administered combination of ethanolic extracts Nelumbo nucifera and

Eichhornia crassipes, whose readings are easily comparable with those of mice administered

standard drug, Diazepam.


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And Standard Drug On Novelty Induced Hypophagia.

S.no Treatment Home cage

(Mean±SEM)

Novel cage

(Mean±SEM)

1 Vehicle 62±3.582 302.3±4.385

2 Nelumbo nucifera 85.75±14.02 300±1.225

3 Eichhornia crassipes 62±4.708 38.25±8.3


Eichhornia crassipes 76±3.582 247±7.45

5 Standard

(Diazepam 10mg/kg) 63.5±1.323 82±5.148

Latency to drink the condensed milk mixture was highly significant in mice administered

ethanolic extracts of Eichhornia crassipes, which indicates high Anti-anxiety property of the

test drug. Latency time was observed to be lower than that of standard drug, Diazepam.



And Standard.

Drug On Evation Test.

S.no Treatment

No. of remaining

mice in box after 5

mins

1 Vehicle 0

2 Nelumbo nucifera 2

3 Eichhornia crassipes 0

4 Nelumbo nucifera + Eichhornia

crassipes 3

5 Standard

(Diazepam 0.5mg/kg) 4


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The ethanolic extracts of Nelumbo nucifera and combination of Nelumbo nucifera and

Eichhornia crassipes produce a significant decrease in exploratory behaviour pattern as

evident from the results evasion tests. The number of mice remaining in the box were high

when compared to Vehicle and mice administered ethanolic extract of Eichhornia crassipes,

suggesting presence of CNS depressant property in Nelumbo nucifera and combination of

Nelumbo nucifera and Eichhornia crassipes test drugs.



And Standard Drug On Caffeine Induced Stereotypic Behavior Test.

S.no Treatment Stereotypic behavior score

5 mins 30 mins 60 mins

1 Control

(Caffeine 40mg/kg) 3±0.0 3±0.0 3±0.0

2 Nelumbo nucifera 2.5±0.2887 1±0.0 0.75±0.4787

3 Eichhornia crassipes 2.75±0.25 1.25±0.25 0.5±0.2887


Eichhornia crassipes 2.25±0.25 1.75±0.25 0.75±0.25

Table 14a.

S.no Treatment Locomotion score

5 mins 30 mins 60 mins

1 Control

(Caffeine 40mg/kg) 5±0.0 5±0.0 5±0.0

2 Nelumbo nucifera 5±0.0 4.25±0.25 1.5±0.2887

3 Eichhornia crassipes 5±0.0 4.25±0.4787 1.25±0.25




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Treatment of mice with ethanolic extracts of Nelumbo nucifera, Eichhornia crassipes and

combination of Nelumbo nucifera and Eichhornia crassipes significantly decreased

stereotypic behavior of mice with time, when compared to control.



And Standard.

Drug On Catalepsy Test.

S.no Treatment Time spent on dowel (Seconds) (Mean±SEM)


1 Vehicle 26±0.4082 53.75±1.181 120.3±0.6292 100.3±0.25

2 Nelumbo nucifera 6.75±0.25 9.75±0.4787 4±0.0 5.25±0.6292

3 Eichhornia crassipes 11±0.4082 20±0.4082 17±0.4082 19.25±0.25


Eichhornia crassipes 6±0.0 20.75±0.25 9.25±0.4787 15±0.4082

5 Standard

(Resperidone 1mg/kg) 4±0.4082 7.25±0.4787 59.5±0.2887 73±1.581

Mice treated with ethanolic extracts of Nelumbo nucifera, Eichhornia crassipes and

combination of Nelumbo nucifera and Eichhornia crassipes had significantly decreased time


896


spent on dowel at all four observation times (30 mins, 60 mins, 90 mins and 120 mins). Time

spent on dowel was lower than that of mice treated with Standard drug, resperidone,

suggesting excellent anti-psychotic property of test drugs, especially that of Nelumbo

nucifera.



And Standard Drug On Antipsychotic Induced Weight Gain

S.no Treatment Body weight in Grams

0 weeks 2 weeks 4 weeks

1 Vehicle 47.5±2.5 47.5±2.5 48.75±3.146

2 Control

(Risperidone 0.5mg/kg) 35±2.041 42.5±1.443 62.5±4.33

3 Nelumbo nucifera 40±4.082 45±2.041 45±2.041

4 Eichhornia crassipes 45±0.2887 42.50±4.330 40±3.536


Eichhornia crassipes 45±5.000 48.75±1.25 50±0.0

Weight gain of mice treated with ethanolic extracts of Nelumbo nucifera, Eichhornia

crassipes and combination of Nelumbo nucifera and Eichhornia crassipes at third

observation (4 weeks) was found to be minimal when compared to that of Control.


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And Standard Drug On Forced Swim Test.

S.no Treatment Immobility time in seconds

(Mean ± SEM)

1 Vehicle 283.5±12.9

2 Nelumbo nucifera 172±3.367

3 Eichhornia crassipes 213±5.066


crassipes 149±3.937

5 Standard (Imipramine 10mg/kg) 191.5±27.49

Immobility time in mice treated with with ethanolic extracts of Nelumbo nucifera, Eichhornia

crassipes and combination of Nelumbo nucifera and Eichhornia crassipes was observed to

have decreased when compared to control. Immobility time decrease was especially

significant in mice treated with ethanolic extracts of Nelumbo nucifera and combination of

Nelumbo nucifera and Eichhornia crassipes which were both lower than that of mice treated

with standard drug, Imipramine, suggesting excellent anti-depressant property of both.



And Standard Drug On Tail Suspension Test

S.no Treatment Immobility time in seconds

(Mean ± SEM)

1 Vehicle 122.8±3.010

2 Nelumbo nucifera 68±1.080



crassipes 52.5±14.93

5 Standard (Imipramine 10mg/kg) 4±0.4082


898


Immobility time in mice treated with with ethanolic extracts of Nelumbo nucifera, Eichhornia

crassipes and combination of Nelumbo nucifera and Eichhornia crassipes was observed to

have significantly decreased when compared to control, suggestive of anti-depressant

property of both extracts and their combination.



And Standard Drug On Step Down Test.

S.no Treatment Step down time in seconds

(Mean ± SEM)

1 Vehicle 82.5±9.845

2 Nelumbo nucifera 48.25±1.493



Eichhornia crassipes 424.5±2.901

5 Standard

(Piracetam 10mg/kg) 402.8±2.626

Mice treated with ethanolic extract Eichhornia crassipes and combination of ethanolic

extracts of Nelumbo nucifera and Eichhornia crassipes were observed to have high latency to


899


step down, when compared to mice treated with vehicle and Standard drug, piracetam.

Especially mice treated with combination of ethanolic extracts of Nelumbo nucifera and

Eichhornia crassipes were observed to have higher latency time to step down than that of

mice treated with standard drug, piracetam, suggestive of memory enhancing property of

combination of ethanolic extracts of Nelumbo nucifera and Eichhornia crassipes drug.



And Standard Drug On Normothermic Animals.

S.no Treatment Temperature in ºC

0 min 30 mins 60 mins

1 Vehicle 36.7±0.1915 36.55±0.1658 36.43±0.04787

2 Nelumbo nucifera 36.75±0.1323 36.15±0.02887 35.78±0.1887

3 Eichhornia crassipes 36.58±0.1887 36.33±0.1652 36.4±0.1581



Minimal or negligent change in body temperature in normothermic mice was observed in

mice treated with ethanolic extract Eichhornia crassipes and combination of ethanolic

extracts of Nelumbo nucifera and Eichhornia crassipes at all three observations (0 mins, 30

mins, 60 mins) suggesting drug safety and indicating that the drugs do not cause any

hyperthermicreactions.

HISTOPATHOLOGICAL RESULTS

Effect of Normal saline on Brain histopathology

Normal brain tissue was observed.


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Fig 1: Microphotograph of brain of mouse treated with Normal saline for one month

Effect of Nelumbo nucifera (ethanolic leaf extract) on Brain histopathology.

Neuronal degeneration was observed with mild oedema.

Fig 2: Microphotograph of brain of mouse treated with Nelumbo nucifera for one

month.

Effect of Eichhornia crassipes (Ethanolic leaf extract) on brain histopathology.

Neuronal degeneration and prominent glial reaction was observed.

Fig 3: Microphotograph of brain of mouse treated with Eichhornia crassipes for one

month


901


Effect of combination of Nelumbo nucifera and Eichhornia crassipes (ethanolic leaf

extracts) on brain histopathology.

Edema of parenchyma with minimal neuronal degeneration was observed.

Fig 4: Microphotograph of brain of mouse treated with combination of Nelumbo

nucifera and Eichhornia crassipes for one month.

Effect of control (Mouse left untreated after induction of Psychoneurosis) on brain

histopathology.

Acute Neuronal degradation was observed.

Fig 5: Microphotograph of brain of mouse left untreated after psychoneurosis induction

for one month.

Histopathological reports suggest mild neuronal impairement in mice brain of Nelumbo

nucifera, Eichhornia crassipes and combinaton of Nelumbo nucifera and Eichhornia

crassipes treated mice, when compared to complete neuronal degradation and damage found

in brain of mouse left untreated after psychoneurosis induction. This suggests that the plant

extracts studied, have neuronal protective properties.


902


4. CONCLUSION

Plant substances continue to serve as viable sources of drugs for the world population and

several plant-based products are in extensive clinical use (Roja & Rao, 2000). This study

paves the way for further attention and research to identify the active compounds

responsible for the plant neuropharmacological activity. Further studies should be undertaken

to elucidate the exact mechanism of action by which extracts exhert their neuropharmacological effect.

Awareness of local community must be enhanced incorporating the traditional knowledge with

scientific findings. Exploitation of the rapidly growing weed, Eichhornia crasipes can be done

on making the different pharmaceutical products, which will be widely available due to the

high availability and rapid growth. Hence, making the 'Best out of waste'.

ACKNOWLEDGEMENTS

This M.Pharmacy thesis is the result of a challenging journey, upon which many people have

contributed and given their support. I cannot thank my parents enough, for showing their

unlimited support and encouragement throughout the ups and downs of this research thesis.

My utmost, devout and sincerest gratitude and appreciation to Mrs. Mehnoor Farheen for

persevering with me as my advisor, guide and friend throughout the time it took me to

complete this research and write the dissertation. She was there for me from start to finish.

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

Sunitha, Shadan Women 's College of Pharmacy, Hyderabad for constant encouragement

throughout the period, which helped me in the successful completion of this project. 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 always finding time from her busy schedule to

answer queries and inspiring me to work my hardest. I must acknowledge as well the many

friends, lab assistants, and librarians who assisted, advised, and supported my research and

writing efforts. Last but not the least; i would like to thank our Lab helpers, Sultan and

Arshad, who were always flexible with their work timings, just to help me through my

research.

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Documents

PHYTOCHEMICAL EVALUATION AND PHARMACOLOGICAL … · PHYTOCHEMICAL EVALUATION AND PHARMACOLOGICAL SCREENING OF ETHANOLIC LEAF EXTRACTS OF EICHHORNIA CRASSIPES AND NELUMBO NUCIFERA