International Journal of Pharmacology, 2010 ISSN 1811-7775

© 2010 Asian Network for Scientific Information

Anti-Inflammatory and Analgesic Activities of Ethanol Extract of Aerial Parts of Justicia gendarussa Burm.

'C. Jothimanivannan, 'R.S. Kumar and Subramanian

'Department of Pharmaceutical Chemistry, Swamy Vivekanandha College of Pharmacy, Elayampalayam, Tiruchengodu-637 205, Namakkal (Dt), Tamilnadu, India department of Pharmaceutical Technology, Anna University Tiruchirapalli, Tiruchirappalli-620 024, Tamilnadu, India

Abstract: The aim of the present study was to evaluate the anti-inflammatory and analgesic activities of the ethanol extract of aerial parts of Justicia gendarussa (EJG) in animal models. The anti-inflammatoiy activity of the extract was evaluated by using carrageenan-induced rat paw edema and cotton pellet granuloma method. The analgesic activity of the extract was evaluated for its central and peripheral pharmacological actions by using Edcfy's hot plate method and acetic acid-induced writhing, respectively. The stucfy was carried out in two differait dose levels of 250 and 500 mg kg"' orally. The EJG did not produce any mortality up to 2000 mg kg"'. EJG at the dose of 500 mg kg"' showed maximum inhibition of 52% in carrageenan-induced paw edema and 45% inhibition in dry weight cotton pellet granuloma formation. Dose dependent increase in latency of response in the hot plate method and 33% inhibition in acetic acid induced writhings in mice were observed with EJG at the dose of 500 mg kg"'. The pharmacological screening of the extract showed significant (p<0.001-0.01) dose-dependent anti-inflammatory activity with good analgesic profile when compared with reference standard. The presence of flavonoids might be responsible for these activities and which are probably mediated via inhibition of various autocoids formation and release.

Key words: Anti-inflammatory, analgesic, Justicia gendarussa, mouse, rats

INTRODUCTION

In Indian system of medicine, a large number of drugs of either herbal or mineral origin have been advocated for various types of diseases and other different unwanted conditions in humans (Brekhman and Dardimov, 1969). Ayurvedic medicines are largely based upon herbal and herbomineral preparations and have specific diagnostic and therapeutic principles (Patwardhan and Hopper, 1992). Inflammation is a disorder involving localized

increase in the number of leukocytes and a variety of

complex mediator molecules (Mantri and Witiak, 1994). Prostaglandins are ubiquitous substances that indicate and modulate cell and tissue responses involved in inflammation. Their biosynthesis has also been implicated in the pathophysiology of cardiovascular diseases, cancer, colonic adenomas and Alzheimer's diseases

(Smith and De Witt, 1995; Lipsky, 1999).
Medicinal plants are believed to be an important

source of new chemical substances witli potential

therapeutic effects (Franthworth, 1988). The research into plants with alleged folkloric use as pain relievers,

anti-inflammatory agents, should therefore, be viewed as a fruitful and logical research strategy in the search for

new analgesic and anti-inflammatory drugs.
Justicia gendarussa Burm. an evergreen scandent

shrub belonging to the family Acanthaceae is commonly known as Vadaikkuthi in Tamil and widely distributed

throughout river beds of Southern India. In traditional medicinal system, different parts of Justicia gendarussa

have been mentioned to be useful in a variety of diseases. The leaves and tender shoots are diaphoretic and used in chronic rheumatism. Fresh leaves are used to treat edema and earache. The plant lias been used by tlie native medical practitioners and tribes to treat various ailments including liver disorders, tumours, inflammation and skin diseases (Kirtikar and Basu, 1993). Woradulayapinij et al. (2005) reported thai Justicia gendarussa has in vitro HIV type 1 reverse transcriptase inhibitory activity. It has been very recently reported that ethanolic and aqueous extracts of leaves of J. gendarussa inhibits the angiogenesis in vitro in dose dependant manner (Umamaheswari et al., 2009) and stem extract of J. gendarussa shows moderate hepatoprotective activity (Krishna etal, 2010).

Corresponding Author: N. Subramanian, Department of Pharmaceutical Technology, Anna University Tiruchirappaili,

Tiruchirappalli-620 024, Tamilnadu, India Tel; +91-431-2407978 Fax:+91-431-2407999 1

However, to our knowledge, there is no scientific

report on the verification of the use of this plant in the treatment of inflammation, fever, tumors and liver

disorders. Therefore, the aim of this study is to evaluate tlie anti-inflammatory and analgesic potential of tlie ethanolic extract of aerial parts of Justicia gendarussa (EJG) in animal models.

MATERIALS AND METHODS

Collection and extraction: Areial parts of Justicia gendarussa were collected in and around Kutralam in Tirunelveli district, Tamil Nadu, India, during the month of May 2007 and authenticated by Botanical Survey of India, Coimbatore, Tamilnadu, India. The aerial parts were shade dried and pulverized The coarse powder was treated with petroleum ether for dewaxing and removal of chlorophyll. Later, it was packed (250 g) in a soxhlet apparatus and subjected to continuous hot

percolation for 8 h using 450 mL of ethanol (95% v/v) as solvent. The extract was concentrated under vacuum and

dried in a dessicator. The percentage yield was found to be4.5% w/w.

Preliminary phytochemical screening: The extract was screened for the presence of various phytochemical

constituents employing standard screening test

(Wagner e/a/., 1984). Conventional protocol for detecting the presence of steroids, alkaloids, tannins, flavonoids,

glycosides, etc., was used.

Animals: Male Wistar albino rats (150-200 g) and Swiss albino mice (20-25 g) were procured from Venkateshwara

Enterprises, Bangalore, Kamataka, India and used

throughout the study. The animals were housed in microlon boxes in a controlled environment (temperature

25L2°C and 12 h dark/light <ycle) witli standard laboratory diet and water ad libitum. The experiments were

performed in accordance with the guidelines established

by the European community for the care and use of

laboratory animals and were approved by Institutional Animal Ethical Committee (lAEC).

Chemicab: Pentazocin(Ranbaxy, India), Aceclofenac and

Aspirin (Micro Lab., India), Carrageenan type HI

(Sigma, St Louis, USA) and acetic acid (Merck Co.) were used in the pharmacological studies.

Acute toxicity studies (LDs,): Acute Oral Toxicity (ACT) of EJG was determined using Swiss albino mice. The animals were fasted for 3 h prior to the experiment and

were administered with single dose of extracts dissolved in 5% gum acacia (doses ranges from 500-2000 mg kg"' at various dose leveb) and observed for mortality up to 48 h (short term toxicity). Based on the short-term toxicity, tlie dose of next animal was determined as per OECD guideline 425. All the animals were also observed for long-term toxicity (14 days). The LDso of the test extract was calculated using AOT 425 software provided by Environmental Protection Agency, USA.

Anti-inflammatory activity
Carrageenan-induced rat paw edema: The rats were

divided into 4 groups (n = 6). The different groups were treated orally with EJG (250 and 500 mg kg"'), aceclofenac

(10 mg kg"') and vehicle control (5% gum acacia, 1 mL 100 g"'). The ethanol extract, standard drug and vehicle control was administered 1 h prior to injection of 0.1 mL of 1 % freshly prepared suspension of carrageenan in normal saline in the right hind paw sub plantar of each rat The paw volume was measured initially and then at 1, 2 and 3 h after the carrageenan injection by using plethysmometer. The anti-inflammatory effect of EJG was

calculated by the following equation:

Anti-inflammatory activity (%) = (I-Vt'Vc) x lOO

where, Vt represents the paw volume in drug treated animals and Vc represents the paw volume of control

groups animak (Winter etal, 1962).

Cotton pellet-induced granuloma: The animals were divided into 4 groups of 6 animals in each group. The rats were anaesthetized and sterile cotton pellets weighing 1 Oil mg were implanted subcutaneously into both sides of the groin region of each rat Group I served as control and received the vehicle (5% gum acacia, 1 mL 100 g"'). EJG at the concentration of 250 and 500 mgkg"' was administered orally to groups 11, III animals for 7 consecutive days from the day of cotton pellet

implantation. Group IV animals received aceclofenac at a dose of 10 mg for the same period On 8th day, the

animals were anaesthetized and the pellets together with the granuloma tissues were carefully removed and made free from extraneous tissues. The wet pellets were

weighed and then dried in an oven at 60°C for 24 h to constant weight, after that the dried pellets were weighed

again. Increment in the dry weight of the pellets was taken as a measure of granuloma formation. The antiproliferative

effect of EJG was compared with control (D'Arcy et at., 1960).

Int. J. Pharmacol, 2010

2

Analgesic activity Hot plate method

The analgesic activity of EJG was assessed using as

described ly hot plate method of Edcfy and Leimbach

(1953). The evaluated parameters were the latency time for

paw licking and jumping responses on exposure to the hot

plate surface which is kept at 55±PC. The animals were

kept in the hot plate until it lifted one of its hind paws. For this method, the mice were divided into 4 groups of

6 animals each. Group I served as control (5% gum acacia, 1 mL 100 g~'), group EI and group III received EJG at a dose of 250 and 500 mg kg"' orally. Group IV received pentazocin at a dose of 5 mg kg"'. All the treatments were given 30 min before the tliermal stimulus and tlie response

was determined at 60,120 and 180 mia

Acetic acid induced writhing test: The writhing test in

mice was carried out using the method of Koster et al.

(1959). The writhes were induced by intraperitoneal

injection of 0.6% v/v acetic acid (80 mg kg"'). Two different doses of EJG (250 and 500 mg kg"') were

administered orally to the group II and group III of 6 animals each. Group I served as control (5% gum acacia, 1 mL 100 g"') and groiqD IV animals received aspirin at a dose of 300 mg kg"'. The extract and standard drug was administered 30 min before chemical stimulus. The number of muscular contractions was counted over a period of 20 min and is expressed as writhing numbers.

Statistical analysis: Values are expressed as MearttSEM. Statistical analysis was performed with one way analysis of variance (ANOVA) followed by Dunnett's test. p-values<0.05 were considered to be statistically significant when compared to control.

RESULTS

Phytochemical screening: Preliminary pl^4ochemical screening of the ethanolic extract revealed the presence of

alkaloids, glycosides, triterpenes, flavonoids and phenolic compounds. Further separation of the specific

phytochemical is in progress.

Acute toxicity studies OL'Dsn): The extract treated animals were observed for mortality up to 48 h (short term toxicity) and for long-term toxicity (14 days). The study indicated that the EJG did not produce any mortality up to

2000 mg kg"'.

Effect of EJG on carrageenan induced rat paw edema: The

result of EJG against carrageenan-induced paw edema is

shown in Table 1. EJG (250 and 500 mg kg"') gave

significant (p<0.001) reduction of rat paw edema at all assessment times in a dose dependent manner. The

extract showed maximum inhibition of 52% at the dose of 500 mg kg"' after 3 h of drug treatment in carrageenan- induced paw edema whereas the standard drug showed

58% of ii^bition.

Effect of EJG on cotton pellet granuloma: The EJG at the

dose of 500 mg kg"' showed 52 and 45% inhibition in the

wet and dry weight cotton pellet granuloma formation

respectively. The effects of EJG and aceclofenac on the

proliferative phase of inflammation are shown in Table 2. A significant (p<0.01) reduction in the weight of the

cotton pellets was observed with EJG at the dose of 250 and 500 mg kg"' compared with the vehicle control treated rats. However, the degree of reduction was less than the effect caused by aceclofenac.

Effect of EJG on hot plate method: The animals pretreated with EJG (250 and 500 mg kg"') showed a dose dependent increase in latency of response in the hot plate method. The increase in the latency responses were significant

(p<0.01) when compared to control. At time interval of 3 h, 250 and 500 mg kg"' EJG effect were found be

decreased remarkably compared with standard drug.

However, their effects are equal during the second hour of experiment. The results are shown in Table 3.

Table 1: Effect of dhanol extract of aerial parts ofJusticia eendarussa on carrageenan induced rat paw edema Paw volume in (mL)

Treatments Dose
Control (Normal saline) lOniLkg"'
Aceclofenac lOmgkg-'
E J G 250mgkg-'
EJG 300 me ke"'
N = 6. •p<0.001 vs. caitrol. Data were analyzed by one w^ ANOVA followed by Duraictt test

Table 2; Effect of ethanoi extract of aerial oarts of Justicia eendarussa on cotton oellet eranuloma Weight of cotton

2h
0.29±0.003 0.14±a008 (48.27%) 0.23±0.010 (20.68%) 0.17±0.008 (41.37%)

Weight of cotton ellets (m

48.62±3.6 23.54±24« 34.42±24» 26.71±21*

3h
0.5±0.006 0.21±0.130*(58%) 0.26±0.006* (48%) 0.24±0.004» (52%)

Percentaae inhibition

Treatments Dose Control (Ntxmal saline) 10 mL kg~' Aceclofenac lOmgkg"'

pellets (ma) (wet) 183.17±14.3 78.25±6.3*

Percentaae inhibition

EJG 230mgkg- 121.16±12.1* EJG 500 ma k- 87.487±7.4»

N = 6. *p<0.01 vs. ccntrol. Data were analyzed by one way ANOVA followed by Dunnett test 3

Int. J. Pharmacol, 2010

1h 0.17±0.02 0.22±0.007

0. lOtO.006 0. 161:0.001 0.12±0.006

0.121:0.005 {91.im) 0.22±0.007 (0%) 0.14±0.011 (36.36%)

Table 3: Effect of etfianol extract of aerial parts oiJusticiagendarussa on theimic stimulus induced i > pain in rats Reaction time (sec)

TreaUnenls Dose
Ccntrol (Norma! saline) 10 mL kg"'
Pentazocin 5 mg kg"'
EJG 250 mg kg"
EJG 500 mg kg"
N = 6. ♦p<0.01 vs. ccntrol. Data were analyzed by one way ANOVA followed by Dunnctt test

Table 4: Effect of ethanol extract of aerial parts of Justiciagendarussa on chemical stimulus induced (writhing test) pain in rats

development of inflammation and EJG extract showed maximum inhibition of 52% at the dose of 500 mg kg"' in

the second phase of development of inflammation. The standard drug, aceclofenac, showed 58% of inhibition in the second phase. Therefore, it is suggested that the mechanism of action of EJG may be related to prostaglandin synthesis inhibition.

The cotton pellet granuloma metliod has been widely

employed to assess the transductive, exudative and

proliferative components of chronic inflammation and is a typical feature of established chronic inflammatory

reactioa The fluid absorbed by the pellet greatly influences the wet weight of the granuloma and dry

weight correlates well with the granuloma of the

granulomatous tissue formed (Olajide et al, 1999,2000). Administration of EJG at the doses of 250 and 500 mg kg"'

significantly reduced the granulomatous tissue formation when compared to control.

It is known that non-steroidal anti-inflammatory

drugs usually do not increase the pain threshold in normal tissues, whereas, local anesthetics and narcotics do (Ferreira et al, 1978). However, the hot plate test was undertaken to verify if EJG would have any central

analgesic effect The results for the group treated with EJG showed significant activity when compared to control

group and nearly equal to the group treated with pentazocin (5 mg kg"'). Hence, it is assumed tliat EJG has significant central analgesic effect.

During the first 30 min of intraperitoneal injection of acetic acid in rats showed high levels of prostaglandins

PGEja and PGF,^ in peritoneal exudates (Deraedt et al, 1980). It is also proved tliat intraperitoneal administration of acetic acid liberates sympathetic nervous system mediators along with prostaglandins (Hokansan, 1978; Duarte et al, 1988). The EJG was effectively inhibiting the acetic acid induced writhings in mice in dose dependent manner. The results were comparable with the group treated with aspirin Hence, we could assume that EJG

may also involve in the peripheral analgesic activity. Preliminaiy phytochemical screening indicated the

presence of flavonoids in EJG. Selected phenolic

compounds and flavonoids were shown to inliibit both the cyclooxygenase and 5-lipoxygenase pathways

(Ferrandiz et al, 1990; Ferrandiz and Alcaraz, 1991). This

No. of writhing ( 2 0(20 mimnit n )

7 9 .789 ±. »1t 2445

A s p i r i n
E J G
EJG
N = 6. •p<0.01 vs. control. Data were analyzed by one way ANOVA followed by Dunnett test

Effect of EJG on acetic acid induced writhing in mice:

Administration of different doses of the extract (250 and 500 mg kg"') decreased the number of writhings in mice

and the effect was foimd to be dose dependent The reduction was statistically significant (p<0.01) when

compared to control. Aspirin showed a 67% inhibition and EJG showed 33% inhibition at the dose of 500 mg kg"' in

acetic acid induced writhing in mice. The results are given in Table A.

DISCUSSION

The most widely used primary test for screening of

anti-inflammatory agents is Carrageenan induced rat paw edema (Winta- et al., 1962). The development of edema in

the paw of the rat after injection of Carrageenan is believed to be biphasic event The initial phase observed

during the first hour is attributed to the release of histamine and serotonin; the second phase is due to the

release of prostaglandin-like substances (Brito and

Antonio, 1998). Based on this, it could be argued that the

suppression of the first phase may be due to inhibition of the release of early mediators, such as histamine and

serotonin and the action in the second phase may be

explained by an inhibition of cyclooj^genase

(Olajide etal, 1999).
Ueno et al (2000) found that the injection of

carrageenan into the rat paw induces the liberation of

bradykinin, which later induces the biosynthesis of

prostaglandins and other autocoids, which are

responsible for the formation of the inflammatory

exudates. Besides, in the carrageenan-induced rat paw

edema model, the production of prostanoids has been

tlirough the serum expression of COX-2 by a positive feedback mechanism (Nantel etal, 1999). The EJG extract

produced minimum inhibition in the initial phase of

T r e a t m e n t s D o s e

Percentage i n h iibnhiitbitoion

C o n t r o l

l O m L k g - '

3 0 0 m g k g - ' 2 5 0 m g k g - ' 500mgkg"'

2 266.. 5±±1 .17 2. *7 2 « 6 26.27. 7± 1. .6 67 ' 7 '

6 6 . 7 9 2 1 . 4 2 3295

5353..5±11.5.85* 8*

Int. J. Pharmacol, 2010

Oil Ih 31i

2.4±0.15 2.32±0.40 2.3±0.4 7.50±0.22* 2.5±0.22 5.30±0.23* 2.6t0.6 6.83±0.30*

2.45±0.16 2.36±0.14 9.72±1.10» 7.84±0.14* 8.06±0.75'* 6.90±1.10* 8.76±0.36» 7.2at0.36*

4

inhibition reduces the release of arachidonic acid

(Yoshimoto et al, 1983). The exact mechanism by which

flavonoids inhibit these en^mes is not clear. Quercetin,

in particular, inhibits both cyclooxygenase and

lipoxygenase activities, thus, diminishing the formation of these anti-inflammatory metabolites (Robak and

Gryglewski, 1996).
The ability of flavonoids to inhibit eicosanoid

biosynthesis has been documented (Damas et al., 1985;

Hertog etal., 1995). Eicosanoids, such as prostaglandins, are involved in various immunological responses

(Moroney et al., 1988) and are the end products of the

cyclooxygenase and lijxjxygenase patliways. Flavonoids also inhibit both cytosolic and membranal

tyrosine kinases which play key roles in the signal transduction pathway that regulates cell proliferation

(Formica and R^elson, 1995). Further, flavonoids are able to inhibit neutrophils degranulation and thereby decrease

the release of arachidonic acid (Hoult et al, 1994; Tordera e/a/., 1994). Thus, the presence of flavonoids in the extract of EJG might be responsible for the anti-inflammatory and analgesic activity in Wister albino

rats and mice.

CONCLUSION

Data obtained in this study indicated that the ethanol extract of aerial parts of Justicia gendarussa possess

anti-inflammatory and analgesic effects. The presence of flavonoids might be responsible for these activities and

which are probably mediated via inhibition of various autocoids formation and release. Further detailed

investigation is underway to determine the exact

phytoconstituents that are responsible for these activities.

ACKNOWLEDGMENT

The authors are thankful to Dr. M. Karunanithi,

Chairman, Swamy Vivekanandha College of Pharmacy,

Tiruchengodu, Tamilnadu, India for providing infrastructural facilities to carry out tliis study.

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