RESEARCH ARTICLE Mamun et.al / IJIPSR / 2 (8), 2014, 1625-1637

Department of Clinical Pharmacology ISSN (online) 2347-2154

Available online: www.ijipsr.com August Issue 1625

EFFECT ON CNS ACTIVITY AND ANALGESIC ACTIVITY OF

PARTHENIUM HYSTEROPHORUS L BY METHANOLIC

EXTRACT AT AERIAL PART IN SWISS ALBINO MICE

1Abdullah Al Mamun*,

2Ahsan Habib,

3Md. Ershad Alam,

4Md. Mahfuzur Rahman,

5 Most. Shamima Parvin

1,2,3Bangladesh University, Department of pharmacy, Mohammodpur,Dhaka-1207,BANGLADESH.

4 Gono Bishwabidyalay, BANGLADESH 5 Stamford University, BANGLADESH

Corresponding Author:

Abdullah Al Mamun

Bangladesh University

Department of pharmacy

Mohammodpur, Dhaka-1207, BANGLADESH

Email: mamunal007@yahoo.com

Phone: +8801712477029

International Journal of Innovative

Pharmaceutical Sciences and Research www.ijipsr.com

Abstract

In the present study, the analgesic and CNS depressant effect of the aerial part of the methanol extract of

Parthenium hysterophorus was investigated. The methanolic extracts of Parthenium hysterophorus were

evaluated for its analgesic activity in mice by using acetic acid-induced writhing, formalin induced Licking test,

hot plate and tail-flick tests and central nervous system (CNS) depressant effect by using rodent behavioral

models, such as hole cross, open field; thiopental sodium induced sleeping time tests and Tail suspension test

for its anti-depressant properties. The results in acetic acid-induced writhing test and formalin induced licking

test for antinociceptive activity, the methanolic extracts at 2.5mg/kg and 5mg/kg dose exhibited significant

(p<0.001) reduction of writhing response and formalin induced licking response in a dose dependent manner. In

Open Field Test and Hole Cross Test , for anti-depressant activity, both dose levels of 2.5mg/kg and 5 mg/kg

exhibited significantly (p<0.001) reduction of movement of mice and also in tail suspension test and thiopental

sodium induced sleep time test, the methanolic extract of Parthenium hysterophorus showed significant

antidepressant activity compared to standard. These results provide in vivo evidence that aerial parts of

Parthenium hysterophorus in general have significant Analgesic and antidepressant effects.

Key words: Analgesic, Anti-depressant properties, Writhing response.

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Department of Clinical Pharmacology ISSN (online) 2347-2154

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INTRODUCTION

Definition of medicinal plants

A considerable number of definitions have been proposed for medicinal plants. According to the

WHO, “A medicinal plant is any plant which, in one or more of its organs, contains substances

that can be used for therapeutic purposes, or which are precursors for chemo-pharmaceutical

Semi-synthesis.” When a plant is designated as ‘medicinal’, it is implied that the said plant is

useful as a drug or therapeutic agent or an active ingredient of a medicinal preparation.

“Medicinal plants may therefore be defined as a group of plants that possess some special

properties or virtues that qualify them as articles of drugs and therapeutic agents, and are used

for medicinal purposes.

“The plants that possess therapeutic properties or exert beneficial pharmacological effect on the

animal body are generally designated as medicinal plant.” (Ghani, 2005)

History of medicinal plant

The earliest known medical document is a 4000-years-old Sumerian clay tablet that recorded

plant remedies for various illnesses. As evident from the papyrus Ebers (written in about 1500

BC), the ancient Egyptians possessed a good knowledge of the medicinal properties of hundreds

of plants. The pun-tsao containing thousands of herbal cures attributed to Shennung, chain’s

legendary emperor who lived 4500 years ago. The earliest mention of the medicinal use of plants

in the Indian subcontinent is found in Rig Veda (4500-1600 BC), which noted that the Indo-

Aryans used the Soma plant as a medicinal agent. The Badianus Manuscript is an illustrated

document that reports the traditional medical knowledge of the Aztecs (Levetin and McMahon

2003).

Development of drugs from medicinal plants

In recent past years more than 20,000 medicinal plants have been identified. Phytochemical and

pharmacological studies of some of these plants have already resulted in the discovery and

development of many important drugs. Some of the glaring examples of such drugs of plant

origin include the following: Anti- malarial drug from Cinchona species, sedative analgesic

drugs from Papaversomniferum.

Development of drugs from medicinal plants is often an elaborate, laborious, time-consuming

and expensive exercise. Careful phytochemical analysis and pharmacological and clinical tests

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Department of Clinical Pharmacology ISSN (online) 2347-2154

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are pre-requisites for developing drugs from medical plants. The stages involved in the

development exercise may be summarised in the following way:

a) Selection and correct identification of the proper medicinal plant and its extraction with a

suitable solvent.

b) Detection of biological activity of the crude extract and establishment of a bioassay system to

permit the identification of the active fraction.

c) Fractionation of the crude extracts by using Physico-chemical procedures, and monitored by

biological tests, identification and separation of active fractions.

d) Isolation of the active constituents by chromatographic or other techniques and purification

of the isolated compounds by repeated chromatography and crystallisation

e) Establishment of the chemical structures of the pure compounds by various Physico-chemical

techniques and determination of their biological activity by various pharmacological and

toxicological tests

f) Production of the drug in appropriate dosage form.

Plant Biography:

Domain: Eukaryota

Kingdom: Plantae

Phylum: Spermatophyta

Class: Magnoliatae

Order: sterales

Family: Asteraceae

Genus: Parthenium

Species: Parthenium hysterophorus L.

Plant Description

Parthenium hysterophorus is a much-branched, short-lived (annual), upright (erect) herbaceous

plant that forms a basalrosette of leaves during the early stage of growth. It usually grows 0.5-1.5

m tall, but can occasionally reach up to 2 m or more in height.

Mature stems are greenish and longitudinally grooved, covered in small stiff hairs (hirsute), and

become much branched at maturity.

The alternately arranged leaves are simple with stalks (petioles) up to 2 cm long and form a

basalrosette during the early stages of growth. The lower leaves are relatively large (3-30 cm

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Department of Clinical Pharmacology ISSN (online) 2347-2154

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long and 2-12 cm wide) and are deeply divided (bi-pinnatifid or bi-pinnatisect). Leaves on the

upper branches decrease in size and are also less divided than the lower leaves. The undersides

of the leaves, and to a lesser degree their upper surfaces, are covered with short, stiff hairs that

lie close to the surface (they are appressedpubescent). Numerous small flower-heads (capitula)

are arranged in clusters at the tips of the branches (in terminalpanicles). Each flower-head

(capitulum) in borne on a stalk (pedicel) 1-8 mm long. These flower-heads (4-5 mm across) are

white or cream in colour and have five tiny 'petals' (rayflorets) 0.3-1 mm long. They also have

numerous (12-60) tiny white flowers (tubularflorets) in the centre and are surrounded by two

rows of small green bracts (an involucre). Colour changes to light brown when seeds are mature

and about to shed. Flowering can occur at any time of the year, but is most common during the

rainy seasons. Five small 'seeds' (achenes) are usually produced in each flower-head (capitulum).

These achenes (1.5-2.5 mm long) consist of a black seed topped with two or three small scales (a

pappus) about 0.5-1 mm long, two straw-coloured papery structures (actually dead

tubularflorets), and a flat bract.

Fig 1: Photographs of P. hysterophorus

MATERIALS AND METHOD

Preparation of Plant Extract for Experiments

Plants Collection

The leaves of Parthenium Hysterophorus L were collected from the Karwan bazar, Dhaka,

Bangladesh. The time of collection was January, 2013 at the daytime.

Drying of the plant sample

The stem and other adulterants were removed at first. Then the leaves were washed with water to

get the fresh sample. Then the collected samples were dried under shade at room temperature for

five days.

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Department of Clinical Pharmacology ISSN (online) 2347-2154

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Grinding of the dried sample

The dried samples were grounded to coarse powder with Blender Machine (NOWAKE, JAPAN)

a and powdered samples were kept in clean closed glass container. During grinding of sample,

the grinder was thoroughly cleaned to avoid contamination with any other substance that was

grounded previously. The dried grinded powder was weighed by rough balance.

Extraction of Dried Powder Sample

150g of the dried powder was taken in a 500ml beaker. Then methanol was added to the powder

with continuous stirring until the powders were soaked properly. Then the mixer was

continuously stirred after few hours, and the beaker was kept for three days. At fourth day the

extract was collected and filtered using a sterilized cotton filter. The volume of the extract was

reduced by using “Rotary Evaporator”. Then this small volume of extract was dried at room

temperature by normal air flow. After drying, 7.69g of dried extract was obtained from 150g of

powder. This extract was used for investigation.

Overall Extraction Process:

The Flow Chart of the extraction process shown below-

Identification of plants/plants part

Collection of plants at suitable time and session

Drying of plant parts in a suitable size

Grinding

Powders were collected and stored in a cool and dry place

Preparation of extracts with methanol

Filtrations were done

Tests were performed to observe the pharmacological effects of the extract.

Fig.2: Flow Chart of the Overall extraction process of Parthenium hysterophorus

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Table 1: Procedure of Chemical Group Test

Sample Test solution Observation Inference

Test for Alkaloids:

# 2 ml solution of the

extract and 0.2 ml of

dilute hydrochloric acid

0.1 ml of Mayer’s

reagent.

Yellowish buff

colored precipitate

was obtained.

Presence of alkaloid

# 2 ml solution of the

extract and 0.2 ml of

dilute hydrochloric acid.

0.1 ml of

Dragendroff’s reagent.

Orange brown

precipitate was

observed.

Presence of alkaloid

# 2 ml solution of the

extract and 0.2 ml of

dilute hydrochloric acid.

0.1 ml of iodine

solution (Wagner’s

reagent).

Reddish brown

precipitate was

obtained.

Presence of alkaloid

# 2 ml solution of the

extract and 0.2 ml of

dilute hydrochloric acid.

0.1 ml of picric acid

solution (Hager’s

reagent).

Yellowish precipitate

was obtained. Presence of alkaloid

Test for Steroids:

# 10 mg extract dissolved

in 1 ml chloroform.

1 ml sulfuric acid.

Chloroform layer

acquired reddish

brown color and acid

layer showed green

fuorescence.

Presence of steroid

Tests for Flavonoids:

# 10 ml of solution of

extract hydrolyzed with

10%sulfuric acid. This

was extracted with ether

and divided into three

portions.

a) 1 ml dilute

Ammonia solution

b)1 ml dilute sodium

carbonate solution

c) 1 ml dilute sodium

hydroxide solution

a) Greenish yellow

color was obtained.

b) Pale yellow color

was obtained.

c) Yellow color was

obtained.

a) Presence of flavonoids

b) Presence of

Flavonoids

c) Presence of

Flavonoids

Tests for Reducing

Sugars:

# 5 ml solution of extract.

5 ml Fehling’s A and

B solution. Boiled for

5 minutes on a boiling

water bath.

Brick red colored

precipitate was not

observed.

Presence of reducing

sugars

# 5 ml solution of extract.

5 ml Benedict’s

reagent and boiled for

5 minutes on a boiling

water bath.

Brick red colored

precipitate was not

observed.

Presence of reducing

sugars

#5 ml solution of extract.

2 drops of 5% alpha-

Naphthol solution

(Freshly prepared and

added 1 ml of sulfuric

acid on the sides of the

test tube.)

Violet colored ring

was not formed at the

junction of two

liquids.

Presence of reducing

sugars

Tests for Tannins:

# 5 ml solution of extract.

1 ml of 10% Lead

acetate solution. Yellow precipitate. Presence of tannins

# 5 ml solution of extract.

1 ml of 10%

potassium dichromate

solution.

Yellowish brown

precipitate was not

obtained.

Presence of tannins

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Test for Glycosides:

small amount of extract

1 ml of water and a

few drops of sodium

hydroxide solution

A yellow color is

formed Presence of glycosides

Test for Saponins

# 1 ml solution of the

extract

Diluted to 20 ml with

distilled water

Shake during 15

minutes and form 1

centimeter foam layer

Presence of saponins

Test for

Carbohydrates

# 2 ml of extract

2 ml of conc. sulfuric

acid

A red or reddish

violet ring is formed Presence of carbohydrate

Test for resins

1 mg of extract

5 to 10 ml of acetic

anhydride and .005 ml

of sulfuric acid

A bright purplish

color, rapidly

changing to violet is

produced

Presence of resins

RESULT AND DISCUSSION

Hot plate Test:

The hot-plate was used to measure the response latencies. It is also called thermal nociception

test method (Eddy and leimbach et al., 1953). In these experiments, the hot plate was maintained

as at 50± 5 0

C. the reaction time was recorded for animals pre-treated with DMSO (10ml/kg 30

min before orally) as control or Methanolic Extract of Perthenium hysterophorus (MEPH)

(2.5mg/kg, and 5mg/kg orally, 30 min before), morphine (5.0 mg/kg intraperitonially, 15 min

before) which was used as positive control group. Animals were placed into the hot plate

chamber and the time of latency was defined as the time period between the zero point, when the

animal was placed on the hot- plate surface, and the time when animal licked its paw or jumped

off to avoid thermal pain. The latency period of response was taken as the index of

antinociception and was determined at pretreatment, 30, 60, 90 and 120 min after the

administration of the test drugs and standard, in order to minimize the damage on the animal

paws; the cut-off time was taken as 20s.

.

Fig. 3: Hot plate Test

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Hot plate Test:

In the hot plate test there were no significant difference in the antinociceptive effect of 2.5mg/kg

and 5 mg/kg MEPH in the Mean ± SEM value for nociception. The antinociceptive effect of

MEPH is dose dependent. MEPH considerably increase the animal reaction time to the heat

stimulus. The increase was significant in all cases beginning from 60 min compared to control (p

< .05) but the dose of 2.5mg/kg p.o. significantly increased the reaction time beginning from 60

min compared to control (p < .01). Morphine (5 mg/kg) markedly increased pain latency (p <

.001) in all case from 30 min.

Table 2: Effect of whole plant extract of Parthenium hysterophorus on Hot plate Test

Treatment Dose Response times (in seconds)

Pretreatment 30 min 60 min 90 min 120 min

Control

(DMSO)

10

ml/kg 4.80±.267 5.31±0.393 7.24±0.275 7.53±0.180 7.76±0.170

Positive

control

(Morphine)

5

mg/kg ٭0.466±14.73 ٭157.±12.95 ٭0.183±11.23 ٭9.49±0.373 268.5.74±

Group I

(Extract)

2.5

mg/kg

7.37±0.711

٭ ٭0.241±11.60 ٭0.220±9.49 ٭0.121±8.56 ٭7.21±0.152

Group II

(Extract)

5

mg/kg ٭6.68±0.441

٭7.58±0.213

٭8.67±0.523

٭237.9.62±

٭12.57±0.588

Each value is presented as the mean ± SEM (n=5). MEPH= methanolic extract of Parthenium

hysterophorus. DMSO= Dimethyl sulfoxide

٭ Dunnett p< .001. Dunnett test as compared to control.

Tail Flick test:

The procedure is based on the observation that the morphine like drugs selectively prolongs the

reaction time of the typical tail withdrawal reflex in mice (D’amour & Smith, 1941). The animals

of the control, positive control and test groups were treated with DMSO (10ml/kg, P.o.),

Morphine (5 mg/kg, i.p.) and test samples at the dose 2.5mg/kg, p.o. and 5 mg/kg. P.o.

respectively. 1 to 2 cm of the tail of the mice was immersed in warm kept constant at 55 0

C. The

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

and the reaction time was recorded as a mean of the next three reading. A latency period of 20 s

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was defined as complete analgesic and the measurement was stopped when the latency period

exceeded to avoid injury to mice. The latent period of the tail- flick response was determined at

pretreatment, 30, 60, 90 and 120 min after the administration of the test drugs and standard.

Fig. 4 Tail Flick test

Tail Immersion test

The tail-immersion test results showed an antinoceptive activity. There were no significant

difference in the antinoceptive effect of 10 and 2.5mg/kg of MEPH when compared with

reference drugs. MEPH at dose of both 2.5mg/kg, 5mg/kg p.o. significantly increase tail flick

latency period beginning from 30 min compared to control (p < .001) and also Morphine (5

mg/kg) markedly increased tail flick latency compared to control (p < .001) in all case from 30

min.

Table 3: Effect of whole plant extract of Parthenium hysterophorus extract on Tail

Immersion test

Treatment Dose Response times (in seconds)

Pretreatment 30 min 60 min 90 min 120 min

Control

(DMSO)

10

ml/kg 1.18±0.34 1.64±0.08 2.75±0.20 2.89±.028 3.24±0.11

Positive

control

(Morphine)

5

mg/kg 1.28±0.13 4.39±0.04* 4.88±0.15* 5.94±0.18* 7.09±0.07*

Group I

(Extract)

2.5

mg/kg 1.17±0.08 2.75±0.12* 4.05±0.15* 4.68±0.14* 5.19±0.15*

Group II

(Extract)

5

mg/kg 1.15±0.02 2.87±0.20* 4.40±0.17* 4.96±0.12* 5.88±0.18*

Each value is presented as the mean ± SEM (n=5). MEPH= methanolic extract of Parthenium

hysterophorus.

.Dunnett p< .001 respectively. Dunnett test as compared to control٭

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Formalin Induced licking test:

The animals were divided into control, positive control and test groups with five mice in each

group. Mice were orally pretreated with MEPH (2.5mg/kg and 5 mg/kg p.o.), and the control

group received similar volume of vehicle (DMSO, 10 ml/kg, p.o.) 60 min prior the nociceptive

agent. Morphine (10 mg/kg. i.p.) was used as the reference drug and administered 15 min before

the nociceptive agent. Animals received 20 µl of a 2.5% formalin solution (0.92% formaldehyde)

made up in saline, injected intraplantarly in the ventral surface of the right hand paw. Animal

were observed from 0 to 05 min (neurogenic phase) and 15-30 min (inflammatory phase) and the

number of licking of the injected paw was counted and considered as indicative of nociception

(Santos ARS et al., 1999 and Santos ARS et al., 1997).

Fig. 5: Half Writhing Given by Mice

Fig. 6: Full Writhing Given by Mice

Formalin Induced licking test

The results show that the methanolic extract from Parthenium hysterophorus(2.5mg/kg and 5

mg/kg orally) caused a significant dose dependent inhibition of both the neurological (0-5 min.)

and inflammatory (15-30 min.) phase of formalin induced licking. However, their

antinociceptive effects were significantly compared to control (p< .001) in both the early and late

phase and also Morphine (5 mg/kg) markedly increased significantly compared to control (p<

.001) in both the early and late phase

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Table 4: Effect of whole plant extract of Parthenium hysterophorus on Formalin Induced

licking test

Treatment Dose

Licking Number

(Mean± SEM) % Inhibition

Early Phase Late Phase Early Phase Late Phase

Control

(DMSO) 10 ml/kg 117.20±4.56 96.60±3.90 00 00

Positive control

(Morphine) 5 mg/kg 39.60±1.44* 00±00* 66.21 100

Group I

(Extract) 2.5mg/kg 88.00±2.50* 7.40±1.03* 24.91 92.33

Group II

(Extract) 5 mg/kg 90.40±2.30* 2.80±.38* 22.86 76.33

Each value is presented as the mean ± SEM (n=5). MEPH= methanolic extract of Parthenium

hysterophorus.

.Dunnett p< .001 respectively. Dunnett test as compared to control٭

Fig. 7: Graphical Presentation of the effect of whole plant extract of Parthenium

hysterophorus extract on Formalin Induced licking test (First phase Paw Licking Number)

Fig. 8: Graphical Presentation of the effect of whole plant extract of Parthenium

hysterophorus extract on Formalin Induced licking test

(Second phase Paw Licking Number)

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CONCLUSION

Based on the result of the present study, it can be concluded that the all parts of crude plant

extracts of Parthenium hysterophorus. Possesses of acetic acid-induced writhing test, induced

Licking test, hot plate tail-flick tests, central nervous system (CNS) depressant test and

remarkable analgesic potential. Various phytochemical constituents like glycoside, alkaloid,

tannin and carbohydrate present in the plant, as evident from phytochemical analysis. The aerial

parts of extracts shown promising analgesic properties were satisfactory compared to respective

standard drugs. At higher dose, notable analgesic activity was observed from Hot Plate and Tail

Flick Test Dose dependant activity was also identified by all the performed pharmacological

investigations.

ACKNOWLEDGEMENT

We are thankful to Ms Sanjida Haque, department of pharmacy, Bangladesh University. We are

especially grateful to ICDDR'B and Department of Pharmacy, Stamford University Bangladesh.

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