6. pharmacological screening 6.1. anti-asthmatic activity...

CHAPTER 6 PHARMACOLOGICAL SCREENING

________________________________________________________________________

Pharmacognostic, Phytochemical and Pharmacological studies on

Clerodendrum splendens. Family- Verbenaceae. 145

6. PHARMACOLOGICAL SCREENING

6.1. Anti-asthmatic activity

6.1.1. Introduction to asthma:

Asthma is a chronic inflammatory disorder of the respiratory disorder of the respiratory

airway, characterized by increased mucus production and airway hyper responsiveness

resulting in decreased air flow, and marked by recurrent episodes of wheezing, coughing

and shortness of breath. It is a multifactorial disease process associated with genetic,

allergic, environmental, infectious, emotional and nutritional components (Miller, 2001).

There are two types of bronchial asthma, extrinsic and intrinsic .In both types the mucous

membrane and the muscle layers of the bronchi become thickened and the mucous glands

enlarge reducing airflow in lower respiratory tract. During an asthmatic attack spasmodic

contraction of bronchial muscle constricts the airway and there is excessive secretion of

thick sticky mucus which further reduces the airway. Inspiration is normal but only

partial expiration is achieved. The lungs become hyper inflated and there is severe

dyspnea and wheezing. The duration of attack usually varies from a few minutes to hours,

and very occasionally, days (status asthmatics). In severe acute attacks the bronchi may

be obstructed by mucus plugs, leading to acute respiratory failure, hypoxia and possibly

death.

Non- specific factors that may precipitate asthma attacks include:

o Cold air

o Cigarette smoking

o Air pollution

o Upper respiratory tract infection

o Emotional stress

o Strenuous exercise

6.1.1.1. Inflammatory cells in pathogenesis of asthma

In asthma, all cells of the airways are involved and become activated. Included are

eosinophil’s, T cells, mast cells, macrophages, epithelial cells, fibroblasts and bronchial

smooth muscle cells. These cells also regulate airway inflammation and initiate the

process of remodeling by the release of cytokines and growth factors (Busse and

Lemanske, 2001).


________________________________________________________________________



6.1.1.2. Inflammatory mediators

Mast cell degranulation releases interleukins, proteases and other enzymes. Several

classes of important mediators, including arachidonic acid (AA) and its metabolites are

derived from cell membrane phospholipids. Once AA is released, it can be broken down

by COX, to form prostaglandin. PGD2 is potent bronchoconstriction agent. PGD2 and

PGD2α are important inflammatory mediators. TXA2 is produced by alveolar

macrophages, fibroblast, epithelial cells, neutrophils, and platelets within the lung. TXA2

have effects, including bronchoconstriction, involvement in the late asthmatic response,

and involvement in the development of airway inflammation and BHR. 5-lipoxigenase

pathway of arachidonic acid breakdown is responsible for production of cysteinyl

leukotrienes (LTC4, LTD4 and LTE4) that constitute the slow-reacting substance of

anaphylaxis. When stimulated, they produce bronchospasm, mucus secretion,

microvascular permeability and airway edema (Bousquet et al., 2005).

6.1.1.3. Amine mediators

6.1.1.3.1 Histamine

Histamine is considered to play major role in asthmatic attacks. It is important for the

immediate bronchoconstriction response. Antigen induced histamine secretion is initiated

by the bridging of the adjacent IgE receptors on the mast cell surface. This causes rise of

cytosol Ca++. Calmodium (CaM) is the intracellular Ca++ receptor and Ca++- CaM

complex through protein kinase takes place that causes protein phosphorylation and the

enzyme activation (Takie et al., 1992).

6.1.1.3.2. Adenosine

It is released by lung tissue in the time of hypoxia such as, after allergen induce

bronchoconstriction. Mast cell also releases adenosine in response to IgE cross- linking

and other stimuli for mast cell activation. The bronchoconstrictor effect of adenosine is

indirect, resulting from the activation of mast cell degranulation, because adenosine

causes histamine release from mast cells (Peter and White, 1984).

6.1.1.4. Lipid- derived mediators

6.1.1.4.1. Leukotriens

Leukotriens are potent lipid mediators produced by arachidonic acid metabolism in cell

or nuclear membrane. Several types of airway cells, including mast cells, eosinophils,


________________________________________________________________________



macrophages, neutrophils and epithelial cells can synthesize leukotrienes in response to

variety of stimuli. Leukotriene is one of the major inflammatory mediators involved in

asthma pathogenesis. Leukotrien-B4, synthesized predominantly by Leukotrien-A4

hydrolase in neutrophils, is an extremely potent activator of neutrophils, causing

aggregation chemotaxis and degranulation (Peter and White, 1984).

6.1.1.4.2. Prostanoids

Prostanoids include PGs and thromboxane (Tx) which are generated from the arachidonic

acid, usually by the action of the cyclooxygenase. In general, PGFs and PGD2 contract

and PGE relax tracheal muscle. Asthmatic individuals are particularly sensitive to PGF2α,

which may cause intense bronchospasm (Tripathi, 2003).

6.2. Animal required for the pharmacological screening

A) Male albino mice (Swiss strain) weighing 25-28 g were used for anti-cataleptic

activity and analgesic activity.

B) Colony-bred adult Wistar rats (150–200 g) were used for anti-inflammatory

activity.

C) Male guinea pigs (250-400 g) were used for evaluating anti-histaminic action of

extracts on isolated ileum and Guinea pig (Dunkey-Hartley strain) of either sex

weighing (250-400 g) were used for Histamine aerosol induced Broncho-

constriction activity.

The animals were housed under standard laboratory conditions, in groups of six each.

The animal had free access to food and water, standard pelleted laboratory animal diet

(Godrej Agrovet Ltd. Mumbai, India) was provided ad libitum during acclimation and

study period. The animals were kept at temperature 20° – 25° C and relative humidity 30

– 70 %. The cycle of 12-hour light and 12 hour dark was maintained. The experimental

protocol was approved by Institutional Animal Ethical Committee.

6.3. Drugs and Chemicals

The following drugs and chemicals were used. Drugs: Clonidine (Unichem, India),

Haloperidol (Sun pharma, India), Pheniramine maleate, Pentazocine lactate injection

(Ranbaxy, India), Paracetamol injection (Heilenlab, India) and Naloxone hydrochloride

injection (Samarth Life Sciences Pvt. Ltd., India), Histamine (Sigma) and

Chlorpheniramine maleate purchased from commercial source purchased from a


________________________________________________________________________



commercial source. Chemicals: Petroleum ether (60°-80° C) AR, chloroform AR,

methanol AR, ethyl acetate AR and Tween 80 AR, , glacial acetic acid AR, Sodium

chloride, potassium chloride, calcium chloride, magnesium chloride, sodium bicarbonate,

sodium dihydrogen phosphate, glucose and distilled water were purchased from PCL,

India.

6.4. Acute toxicity study

Procedure

Acute toxicity study of petroleum ether extract of stem, chloroform extract of stem,

methanol extract of stem, petroleum ether extract of flowers, methanol extract of flowers,

petroleum ether extract of leaves, ethyl acetate extract of leaves, methanol extract of

leaves of Clerodendrum splendens were performed in adult albino mice of either sex.

Animals were divided into various groups (n=6 in each group). The mice were fasted for

18 hr and above mentioned extracts were administered in six different doses: 500, 800,

900, 1000, 1100, 1200 and 1300 (mg/kg, i.p) to individual groups. Control group mice

received 5% Tween 80 solution in distilled water. All the mice were observed for 72 hr

and the LD50 was calculated (Turner, 1971; Harish et al., 2001).

6.4.1. Result and Discussion

The LD 50 values of petroleum ether extract of stem, chloroform extract of stem,

methanol extract of stem, petroleum ether extract of flowers, methanol extract of flowers,

petroleum ether extract of leaves, ethyl acetate extract of leaves, methanol extract of

leaves of Clerodendrum splendens when given intraperitoneally and tested in albino mice

was found to be more than 1300 mg/kg body weight. Based on the result of preliminary

toxicity testing and published literature on Clerodendrum genus the dose of 50 and 100

mg/kg (p.o.) were chosen for In-vivo histamine aerosol induced bronchoconstriction in

Guinea pigs studies (Stephen et. al., 2012, Bhujbal et. al. 2009).

The doses for anti-cataleptic activity, analgesic activity and anti-inflammatory activity

was decided based on observed result of In-vivo bronchoconstriction experiments in

Guinea pig for C. splendens plant extract.

The route of administration of C. splendens extracts and standard drugs used for In-vivo

studies were as per published literature selected as guide for performing the experiments.


________________________________________________________________________



6.5. Assessment of Anti-asthmatic activity

6.5.1. Studies on smooth muscle preparation of guinea pig ileum (In-vitro)

Procedure:

Paranajape and Mehta, 2008; Bhujbal et. al., 2009 and Nirmal et. al., (2012) have

described this method. Guniea pigs were sacrificed by a sharp blow over the head,

abdomen was opened, ileum was dissected out and a small piece of 2-3 cm was taken

from portion situated 15 cm proximal in the ileo-caecal junction and suspended in Tyrode

solution (NaCl 8.0, KCl 0.2, CaCl2 0.2, MgCl2 0.1, NaHCO3 1.0, NaH2PO4 0.05 and

glucose 1 gm/lit) and continuously aerated at 37° ± 0.5° C. The tissue was allowed to

equilibrate for 30 min under a load of 500 mg. After equilibration period a contact time

of 30 sec and 5 min time cycle was followed for recording the responses of histamine by

using frontal writing lever. After obtaining a dose response curve of histamine on ileum,

petroleum ether extract (80 μg/ml) of C. splendens stem was added to the reservoir

containing Tyrode solution and a response of tissue was recorded for the same dose of

histamine (0.1, 0.2, 0.4,0.8 and 1.6 ml) to obtain inhibition curve. After several washings,

chloroform, methanol extract of stem, petroleum and methanol extract of flower and

petroleum ether, ethyl acetate and methanol extract of leaves (80 μg/ml) of C. splendens

was added to the reservoir containing Tyrode solution and same procedure was followed

to obtain inhibition curve separately.

Graph of percentage of maximum relaxation response versus concentration of histamine

was plotted to record dose response curve of histamine in presence of various extracts of

C. splendens stem, flower and leaves.


Histamine (0.1-1.6 ml) causes contraction of guinea pig ileum. Ethyl acetate extract of

leaves and petroleum ether extract of stem (80 μg/ml) of C. splendens resisted contraction

induced by various concentrations of histamine (0.1-1.6 ml) better than other extracts.

(Table 54, Figure 91-92).

Guinea pig ileum is used for screening of anti-histaminic activity. The stimulation of H1

receptors produces graded dose related contraction of isolated guinea pig ileum (Saraf et.

al., 1998). In the present study, ethyl acetate extract of leaves and petroleum ether extract

of stem of C. splendens (80 μg/ml) significantly inhibited the histamine induced


________________________________________________________________________



contraction of isolated guinea-pig ileum preparation indicating its H1 receptor

antagonistic activity and involvement of β2-agonists on the relaxation of the tissue. The

ability of the ethyl acetate extract of leaves and petroleum ether extract of stem to inhibit

the contraction induced by the bronchoconstrictor histamine suggests a possible role in

the treatment of asthma and supports the anti-asthmatic properties of the plant.

Table 54: Effect of various extracts of Clerodendrum splendens (80 µg/ml) on histamine

-induced contraction on guinea pig ileum.

% inhibition of smooth muscle contraction

Dose in ml of

histamine (80 μg/ml) PE-S CL-S MT-S PE-L EA-L MT-L PE-F MT-F

0.1 68.42 50.00 52.63 53.16 76.32 55.26 57.89 52.63

0.2 60.53 34.21 44.74 42.63 71.05 44.74 39.47 36.84

0.4 55.26 21.05 34.21 37.37 65.79 34.21 28.95 26.32

0.8 50.00 7.89 21.05 28.47 63.16 15.79 23.68 18.42

1.6 47.37 0.00 10.53 24.21 60.53 7.89 18.42 13.16

Where, PE-S – Petroleum ether extract of stem; CL-S – Chloroform extract of stem; MT-

S – Methanol extract of stem; PE-F- Petroleum ether extract of flowers; MT-F- Methanol

extract of flowers; PE-L – Petroleum ether extract of leaves; EA-L – Ethyl acetate extract

of leaves; MT-L – Methanol extract of leaves.

Figure 91: Effect of various extracts of C. splendens (80 µg/ml) on histamine-induced

contraction on guinea pig ileum.


________________________________________________________________________



Figure 92: Effect of various extracts of C. splendens (80 µg/ml) on histamine-induced

contraction on guinea pig ileum.

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

0.1 0.2 0.4 0.8 1.6

% I

nh

ibit

ion

of

smo

oth

mu

scle

con

tra

ctio

n

Histamine (µg/ml)

PE-S

CL-S

MT-S

PE-L

EA-L

MT-L

PE-F

MT-F

6.5.3. Histamine aerosol induced bronchoconstriction in Guinea pigs:

This method is been used to assess direct anti-histaminic activity of plant extract. The

experiment was done to examine the In-vivo model for Histamine induced bronchospasm

in Guinea pig.

Procedure

Stephen et. al., 2012; Saxena 2014 have described this method. Histamine hydrochloride

was dissolved in distilled water to prepare 0.2% w/v solution. Experimentally bronchial

asthma was induced in guinea pigs by exposing histamine aerosol using nebulizer with

constant pressure 40mm/Hg in an aerosol chamber (30 x 15 x 15cm) made of Perspex

glass. The required time for appearance of preconvulsive dyspnoea produced by the


________________________________________________________________________



histamine was noted for each animal. Each animal was placed in the histamine chamber

and exposed to 0.2 % histamine aerosol. The preconvulsion time (PCT), i.e. the time of

aerosol exposure to the start of dyspnoea leading to the appearance of convulsion, was

noted. As quickly as the preconvulsion dyspnoea (PCD) was recorded, the animals were

removed from the chamber and positioned in fresh air for recover. This time for

preconvulsive dyspnoea was recorded as basal value. Guinea pigs were then allowed to

recover from dyspnoea for 2 days. After that, the animals were allotted to different

groups of 6 animals per group. Animals in group 1 served as control and received

distilled water. The animals of groups 2 to 9 were given each, by oral intubation, 50

mg/kg and animal of groups 10- 17 were given each, by oral intubation 100 mg/kg dose

of petroleum ether extract of stem, Chloroform extract of stem, Methanol extract of stem,

Petroleum ether extract of flowers, Methanol extract of flowers, Petroleum ether extract

of leaves, Ethyl acetate extract of leaves and Methanol extract of leaves of

Clerodendrum splendens respectively, while group 18 received the standard drug -

Chlorpheniramine maleate, 1 mg/kg intraperitoneally. After receiving the drugs, all the

animals were again exposed to histamine aerosol in the chamber, one hour, four hours

and 24 hrs, to determine pre convulsive time (PCT). The protection untaken by the

treatment was calculated using the formula

Percentage protection = 1 – T1 x 100 (% Protection offered by the extract)

T2

Where: T1 is the mean of PCT (preconvulsion time) before administration of test drugs.

T2 is the mean of PCT (preconvulsion time) after administration of test drugs at 1 hr, 4

hrs and 24 hrs.


The ethyl acetate extract of leaves and petroleum ether extract of stem of C. splendens

expressively extended the latent period of spams followed by exposing to histamine

aerosol at the dose of 50 mg/kg, (p.o.) which showed extreme protection of 65.80% and

58.82% at the time 4 hour as compared to Chlorpheniramine maleate (standard) 1 mg/kg,

i.p. which undertaken maximum protection of 75.46% at time 4 hours (Table 55, 56 and

Figure 93, 95).


________________________________________________________________________



The ethyl acetate extract of leaves and petroleum ether extract of stem of C. splendens

expressively extended the latent period of spams followed by exposing to histamine

aerosol at the dose of 100 mg/kg, (p.o.) which showed extreme protection of 67.18% and

59.31% at the time 4 hour as compared to Chlorpheniramine maleate (standard) 1 mg/kg,

i.p. which undertaken maximum protection of 75.46% at time 4 hours (Table 55, 57 and

Figure 94, 96).

The results obtained in above study indicates no significant protection at 100 mg/kg

(67.18% & 59.31% ) dose compared to 50 mg/kg (65.80% & 58.82%) dose level of the

ethyl acetate extract of leaves and petroleum ether extract of stem of C. splendens.

Hence, for anti-cataleptic activity, analgesic activity and anti-inflammatory activity

studies dose level of 50 mg/kg was selected.

Asthma, the atopic disease with the greatest clinical and economic effect is an allergic

and inflammatory outward sign of respiratory disorders. It is essentially characterized by

the restriction of tracheal muscle obstruction (Barnes, 2000). The syndrome of bronchial

asthma is characterized by wide spread narrowing of the bronchial tree due to contraction

of the smooth muscle in response to multiple stimuli resulting in the release of chemical

mediators such as Histamine (Abraham et. al., 1992).

Histamine induced bronchoconstriction is the traditional immunological model of antigen

induced airway obstruction. Histamine when inhaled causes hypoxia and leads to

convulsion in guinea pigs and causes very strong smooth muscle contraction, profound

hypotension, and capillary dilation in cardiovascular system. A prominent effect caused

by histamine leads to severe bronchoconstriction in the guinea pigs that causes asphyxia

and death. Bronchodilators can delay the occurrence of these symptoms (Sunanda et. al,

1981).

The results of the study thus confirmed the bronchodilator properties of the plant,

justifying its traditional claim in the treatment of asthma. Drugs effective in the asthma

are mostly steroidal in nature (Bouic, 1999). Phytochemical profile of the C. splendens

plant reveals the presence of steroid, terpenoids and various flavonoids compounds.

Current study resolved that Clerodendrum splendens ethyl acetate of leaves and

petroleum ether extract of stem possess highly substantial anti-asthmatic activity by

significantly inhibited the histamine induced bronchoconstriction of guinea pig


________________________________________________________________________



representing its H1 receptor antagonistic activity and support the plants by its anti-

asthmatic properties.

Table 55: Effect of various extracts of Clerodendrum splendens (50 &100 mg/kg, p.o.)

against Histamine induced bronchoconstriction in guinea pigs.

Treatment Dose

(mg/kg)

Latent period of convulsion (sec) Mean ± SEM

Before 1 hr 4 hrs 24 hrs

Control --- 15.45 ± 0.72 14.95 ± 0.31 15.65 ± 1.25 15.98 ± 0.91

CHM 1mg/kg 15.15 ± 0.87 54.65 ± 2.46* 61.73 ± 2.37* 27.58 ± 1.03*

PE-S 50 mg/kg 15.67 ± 0.53 30.31 ± 0.92* 38.05 ± 0.24* 23.44± 0.80*

PE-S 100 mg/kg 16.53 ± 0.37 35.88 ± 0.97* 40.62 ± 1.09* 23.80± 0.94*

CL-S 50 mg/kg 15.85 ± 0.60 20.49 ± 1.28 16.67 ± 0.98 15.90 ± 0.55

CL-S 100 mg/kg 15.40 ± 0.92 21.60 ± 1.23 17.38 ± 1.09 15.60 ± 1.07

MT-S 50 mg/kg 15.49 ± 0.86 23.08 ± 0.68 25.46 ± 0.73* 17.69 ± 0.45

MT-S 100 mg/kg 16.33 ± 0.82 23.88 ± 0.64* 26.35 ± 0.72* 18.27 ± 0.40

PE-F 50 mg/kg 15.75 ± 0.84 22.10 ± 0.63 24.60 ± 0.80* 17.78 ± 0.72

PE-F 100 mg/kg 15.55 ± 1.11 23.17 ± 0.58 25.53 ± 0.80* 18.45 ± 0.63

MT-F 50 mg/kg 16.73 ± 0.89 20.65 ± 1.26 24.54 ± 0.47* 16.81 ± 0.82

MT-F 100 mg/kg 16.43 ± 0.79 21.88 ± 1.19 26.53 ± 0.29* 17.43 ± 0.92

PE-L 50 mg/kg 15.85 ± 1.02 26.32 ± 1.16* 27.08 ± 0.43* 17.73 ± 1.29

PE-L 100 mg/kg 14.87 ± 1.04 27.82 ± 0.81* 30.10 ± 0.77* 19.72 ± 1.54

EA-L 50 mg/kg 15.93 ± 1.13 37.32 ± 1.26* 46.58 ± 2.04* 23.71 ± 1.33*

EA-L 100 mg/kg 15.83 ± 0.73 41.48 ± 1.60* 48.23 ± 1.83* 24.55 ± 1.32*

MT-L 50 mg/kg 16.58 ± 0.95 19.89 ± 0.57 17.74 ± 1.37 16.72 ± 0.50

MT-L 100 mg/kg 16.43 ± 0.90 20.60 ± 0.62 18.28 ± 1.39 16.62 ± 0.43


________________________________________________________________________



All the data are expressed as mean ± SEM, n=six in each group. *P<0.05 compared to

control (One way ANOVA followed by Dunnett’s test).

Statistical Analysis

The data is presented as mean ± SEM. The data was analyzed by one-way ANOVA

followed by Dunnett’s test. Prism Graph pad 3 was used for statistical analysis. P<0.05

was considered significant.

Table 56: Percentage protection of Clerodendrum splendens (50 mg/kg, p.o.) against

Histamine induced bronchoconstriction in guinea pigs.

Treatment Dose

(mg/kg)

Percentage Protection (%)

1 hr 4 hrs 24 hrs

CHM 1 72.28 75.46 45.07

PE-S 50 48.30 58.82 30.17

CL-S 50 22.65 4.92 0.31

MT-S 50 32.89 39.16 12.44

PE-F 50 28.73 35.98 11.42

MT-F 50 18.98 31.83 0.42

PE-L 50 39.78 41.47 10.60

EA-L 50 57.32 65.80 32.81

MT-L 50 16.64 6.54 0.72

Table 57: Percentage protection of Clerodendrum splendens (100 mg/kg, p.o.) against

Histamine induced bronchoconstriction in guinea pigs.

Treatment Dose

(mg/kg)

Percentage Protection (%)

1 hr 4 hrs 24 hrs

CHM 1 72.28 75.46 45.07

PE-S 100 53.93 59.31 29.06

CL-S 100 28.70 11.39 1.28


________________________________________________________________________



MT-S 100 31.62 38.03 10.62

PE-F 100 32.89 39.09 15.72

MT-F 100 24.91 38.07 5.74

PE-L 100 46.55 50.60 24.59

EA-L 100 61.84 67.18 35.52

MT-L 100 20.24 10.12 1.14

Where, CHM –Chlorpheniramine maleate; PE-S – Petroleum ether extract of stem; CL-S

– Chloroform extract of stem; MT-S – Methanol extract of stem; PE-F- Petroleum ether

extract of flowers; MT-F- Methanol extract of flowers; PE-L – Petroleum ether extract of

leaves; EA-L – Ethyl acetate extract of leaves; MT-L – Methanol extract of leaves.

Figure 93: Effect of various extracts of C. splendens (50 mg/kg, p.o.) on histamine

induced bronchoconstriction in guinea pigs


________________________________________________________________________



Figure 94: Effect of various extracts of C. splendens (100 mg/kg, p.o.) on histamine

induced bronchoconstriction in guinea pigs

Figure 95: Percentage protection of various extracts of C. splendens (50 mg/kg, p.o.) on

histamine induced bronchoconstriction in guinea pigs


________________________________________________________________________



Figure 96: Percentage protection of various extracts of C. splendens (100 mg/kg, p.o.) on

histamine induced bronchoconstriction in guinea pigs

Where, CHM –Chlorpheniramine maleate; PE-S – Petroleum ether extract of stem; CL-S

– Chloroform extract of stem; MT-S – Methanol extract of stem; PE-F- Petroleum ether



6.6. Assessment of Anti-cataleptic activity

Catalepsy is a condition in which the animal maintains imposed posture for long time

before regaining normal posture. Catalepsy is a sign of extra-pyramidal effect of drugs

that inhibit dopaminergic transmission or increase histamine release in brain. Clonidine, a

α2-adrenoceptor agonist, induces dose dependent catalepsy in mice, which is inhibited by

histamine H1 receptor antagonists but not by H2 receptor antagonist (Jadhav et al., 1983).

They also showed that pretreatment with L-histidine, a precursor of histamine potentiated

clonidine-induced catalepsy in dose dependent manner. Muley et al., (1979) showed that

intracerebroventricular injection of histamine in conscious mice induced catalepsy, which


________________________________________________________________________



was inhibited by H1 receptor antagonist but not by H2 receptor antagonist. It is known that

clonidine releases histamine from mast cells (Lakadwala et al., 1980). Schwatz (1997)

identified histamine containing mast cells in brain. Clonidine-induced release of

histamine from mast cells is inhibited by α2-adrenoceptor blocker, prazocine (Gilman and

Goodman, 1985). Neuroleptic agent also induced catalepsy, but by different mechanism.

Neuroleptic agents inhibit dopamine D2 receptor in the substantia nigra (Sanberg, 1980;

Ossowska et al., 1990). C. splendens is used for the treatment of asthma in folk medicine

hence it was our objective to study the effect its stem, leaves and flower extracts

scientifically on clonidine-induced catalepsy (Kirtikar and Basu, 1988). Since catalepsy is

a common extra-pyramidal side effect of neuroleptic agents and the effect of the plant on

haloperidol-induced catalepsy is not known, we also studied their effect on haloperidol-

induced catalepsy in mice.

6.6.1. Effect on Clonidine-Induced Catalepsy

Procedure

Bar test was used to study the effect of various extracts of stem, flower and leaves on

Clonidine-induced catalepsy (Ferre et al., 1990; Nirmal et al., 2011). Clonidine (1 mg/kg,

s.c.) was injected to mice (n = 6) pretreated 30 min before with vehicle (5 ml/kg, i.p.),

Petroleum ether extract of stem, Chloroform extract of stem, Methanol extract of stem,

Petroleum ether extract of flowers, Methanol extract of flowers, Petroleum ether extract

of leaves, Ethyl acetate extract of leaves, Methanol extract of leaves of Clerodendrum

splendens (50 mg/kg, i.p., each) and pheniramine maleate (10 mg/kg, i.p.). The forepaws

of mice were placed on horizontal bar (1 cm in diameter, 3 cm above the table) and the

time required to remove the paws from bar was noted for each animal and the durations

of catalepsy was measured at 0, 15, 30, 60, 90, 120, 150 and 180 min

6.6.2. Result for Clonidine-Induced Catalepsy

Clonidine (1 mg/kg, s.c.) is used for induction of catalepsy in mice. Clonidine induced

duration of catalepsy in mice up to 402 ± 0.2 sec. Ethyl acetate extract of leaves and

petroleum ether extract of stem of C. splendens inhibited clonidine-induced catalepsy

significantly better than other extracts. Activity of extracts was compared with standard

drug pheniramine maleate (10 mg/kg, i.p.). (Table 58 and Figure 97).


________________________________________________________________________



Table 58: Effect of various extracts of Clerodendrum splendens (50 mg/kg, i.p.) on

Clonidine-Induced Catalepsy in mice.

Treatment Dose

(mg/kg)

Duration of catalepsy (sec) Mean ± SEM

0 min 30 min 60 min 90 min 120

min

150

min

180

min

Control - 27±0.7 175±0.4 281±0.3 347±0.4 392±0.2 398±0.1 402±0.2

PHM 10 27±0.4 50±0.7* 82±0.7* 58±0.2* 40±0.4* 31±0.2* 28±0.2*

PE-S 50 25±0.2 59±0.3* 116±0.2* 87±0.3* 62±0.3* 49±0.2* 36±0.3*

CL-S 50 25±0.4 180±0.1 275±0.1 307±0.3 297±0.2 268±0.2 251±0.2

MT-S 50 25±0.1 80±0.2 129±0.1* 101±0.3 97±0.2* 68±0.2* 51±0.2*

PE-F 50 21±0.7 72±0.3* 128±0.4* 97±0.4* 71±0.4* 52±0.5* 37±0.1*

MT-F 50 22±0.6 85±0.4 145±0.5 147±0.6 101±0.3 98±0.7* 52±0.7*

PE-L 50 25±0.8 69±0.2* 122±0.6* 92±0.7* 68±0.2* 51±0.6* 37±0.4*

EA-L 50 21±0.3 58±0.3* 111±0.7* 78±0.5* 67±0.1* 48±0.7* 29±0.7*

MT-L 50 21±0.4 87±0.2 152±0.3 165±0.4 158±0.7 97±0.3* 53±0.5*



Where, PHM –Pheniramine maleate; PE-S – Petroleum ether extract of stem; CL-S –

Chloroform extract of stem; MT-S – Methanol extract of stem; PE-F- Petroleum ether








________________________________________________________________________



Figure 97: Effect of various extracts of C. splendens (50 mg/kg, i.p.) on Clonidine-

Induced Catalepsy in mice.

0 30 60 90 120 150 1800

100

200

300

400

500Control

PHM

PE-S

CL-S

MT-S

PE-F

MT-F

PE-L

EA-L

MT-L

**

** * *

**

**

*

Time interval (min)

Dura

tion o

f cata

lepsy (

sec)

Mea

n ±

SE

M



Where, PHM –Pheniramine maleate; PE-S – Petroleum ether extract of stem; CL-S –

Chloroform extract of stem; MT-S – Methanol extract of stem; PE-F- Petroleum ether



6.6.3. Effect on Haloperidol-Induced Catalepsy

Procedure

The same Bar test was used using haloperidol (Ferre et al., 1990; Nirmal et al., 2011).

Haloperidol (1 mg/kg, i.p.) was injected to mice (n = 6) pretreated 30 min before with

vehicle (5 ml/kg, i.p.), petroleum ether extract of stem, Chloroform extract of stem,

Methanol extract of stem, Petroleum ether extract of flowers, Methanol extract of

flowers, Petroleum ether extract of leaves, Ethyl acetate extract of leaves, Methanol

extract of leaves of C. splendens (50 mg/kg, i.p., each). The durations of catalepsy was

measured at 0, 15, 30, 60, 90, 120, 150 and 180 mins.


________________________________________________________________________



6.6.4. Result for Haloperidol-Induced Catalepsy

None of the extracts of C. splendens inhibited haloperidol induced catalepsy significantly

(Table 59 and Figure 98)


Haloperidol-Induced Catalepsy in mice.

Treatment Dose

(mg/kg)

Duration of catalepsy (sec)

Mean ± SEM

0 min 30 min 60 min 90 min 120

min

150

min

180

min

Control - 27±0.7 175±0.4 281±0.3 347±0.4 392±0.2 398±0.1 402±0.2

PE-S 50 23±0.2 160±0.3 269±0.2 330±0.6 380±0.5 381±0.2 391±0.3

CL-S 50 25±0.1 171±0.2 278±0.1 332±0.3 382±0.2 378±0.2 391±0.2

MT-S 50 24±0.3 165±0.2 268±0.5 331±0.4 376±0.3 375±0.1 396±0.4

PE-F 50 22±0.6 161±0.4 267±0.5 332±0.6 376±0.3 384±0.7 389±0.7

MT-F 50 23±0.2 162±0.3 271±0.4 336±0.5 379±0.2 379±0.3 382±0.1

PE-L 50 19±0.3 164±0.3 271±0.7 327±0.5 364±0.1 374±0.7 385±0.7

EA-L 50 25±0.4 169±0.2 275±0.3 329±0.4 381±0.2 390±0.1 379±0.5

MT-L 50 23±0.7 162±0.1 271±0.7 315±0.6 351±0.6 372±0.7 385±0.6












________________________________________________________________________



Figure 98: Effect of various extracts of C. splendens (50 mg/kg, i.p.) on Haloperidol-

Induced Catalepsy in mice.

0 30 60 90 120 150 1800

100

200

300

400

500Control

PE-S

CL-S

MT-S

PE-F

MT-F

PE-L

EA-L

MT-L

Time interval (min)

Du

rati

on o

f cata

lep

sy

(sec)

Mean ±

SE

M







6.6.5. Discussion for Anti-cataleptic activity

Several drugs are known to induce catalepsy in animals. The neuroleptic agents induce

catalepsy by inducing dopamine D2 receptor in the substantia nigra (Sanberg, 1980).

Chopra and Dandiya (1975) have studied the relative role of acetylcholine and histamine

in perphenazine-induce catalepsy and suggested that anticholinergic activity of

antidepressant might be due to an increase in dopamine content in brain or their ability to

inhibit release of acetylcholine. They also showed that different stages of catalepsy

appear to be directly correlated with brain histamine content. Uvnas (1969) studied the

mast cell degranulation and its correlation with the release of histamine after

administration of mast cell degranulating agent (Compound 48/80). Lakdawala et al.,


________________________________________________________________________



(1980) have shown that clonidine releases histamine from mast cell in a similar manner to

a selective liberator like compound 48/80. The observation of this study indicated that the

plant C. splendens having anti-histaminic activity inhibited clonidine-induced catalepsy

and not inhibited haloperidol-induced catalepsy. From the present study we can conclude

that the cataleptic effect of clonidine in the mouse is mediated by histamine release from

mast cells. The effect of this extracts on clonidine-induced catalepsy is probably due to

their mast cell stabilizing property and the plant does not have activity on dopaminergic

transmission. It can be concluded that the constituents present in ethyl acetate extract of

the leaves of C. splendens may be useful as anti-histaminic and may be used in the

treatment of asthma.


________________________________________________________________________



6.7. Analgesic and Anti-flammatory activity

Traditionally the plant is used for the treatment of asthma. Inflammation of lungs and

airways is one of the symptoms in asthma. Anti-inflammatory and analgesic drugs play

an significant role in the treatment of asthma by providing relief from the inflammation

and pain of the airways. Hence, objective of the present study was to check the analgesic

and anti-inflammatory potential of plant.

6.7.1. Assessment of Analgesic activity by Hot plate test

Procedure:

Central nociceptive activity was evaluated using the hot plate method (Woolfe and

MacDonald, 1944; Nirmal et al., 2012). Mice were divided into 10 groups of six animals

each. The first group served as control and received only vehicle (10% Tween 80 in

distilled water), and the second group was administered standard drug pentazocine (10

mg/kg, i.p.). The animals of third to tenth groups were treated with petroleum ether

extract of leaves, ethyl acetate extract of leaves, methanol extract of leaves, petroleum

ether extract of stem, chloroform extract of stem, methanol extract of stem, petroleum

ether extract of flower and methanol extract of flower of Clerodendrum splendens (50

mg/kg, i.p., each), respectively. All the extracts and standard drug were dissolved into

the vehicle. The mice were placed individually on the hot plate maintained at 55 ± 0.2oC,

and latency of nociceptive response such as licking, flicking of the hind limbs or jumping

was noted. The readings were taken at 0, 30, 60, 90, 120 and 150 min after treatment. The

experiment was terminated 20 sec after their placement on the hot plate to avoid damage

to the paws.

6.7.2. Results and Discussion

Ethyl acetate extract (50 mg/kg, i.p.) of leaves of Clerodendrum splendens showed

significant increase in reaction time as compared to control and other extracts. It showed

promising activity comparable to the standard drug pentazocine (10 mg/kg, i.p.).

Petroleum ether extract of leaves, stem and flowers also showed significant activity

(Table 60 and Figure 99).

Thermal stimuli causes pain by stimulation of nociceptive receptors and transmitted over

intact neural pathways. The hot plate test is the specific central antinociceptive test

(Parkhouse and Pleuvry, 1979). The thermal stimulus is also described as an acute, non-


________________________________________________________________________



inflammatory nociceptive stimulus as it causes direct stimulation of the nociceptors

without causing any inflammatory mediated nociception. Ethyl acetate extract (50 mg/kg,

i.p.) of leaves of Clerodendrum splendens showed significant results in this test, so there

may be involvement of opioid receptors. The analgesic activity was significant to

pentazocine, an opioid analgesic whose analgesic activity is mediated through central

route. The analgesic effect of opioids arise from their ability to directly inhibit the

ascending transmission of nociceptive information from the spinal cord dorsal horn and

to activate pain control circuits that descends from the mid brain via the rostral

ventromedial medulla to the spinal cord dorsal horn. The opioid agents exert their

analgesic action via the supraspinal (µ1, κ3, δ1, σ2) and spinal (µ2, κ1, δ2) receptors

(Reisine and Pasternack, 1996).


thermic stimulus-induced pain (Hot plate test).

Treatment

Reaction time (Sec)

Time after treatment (min)

0 30 60 90 120 150

Control 2.3±0.3 2.8±0.2 2.4±0.3 2.6±0.4 2.1±0.1 2.8±0.2

Pentazocine (10

mg/kg, i.p.) 2.8±0.4 7.4±0.3* 11.9±0.4* 10.2±0.3* 6.1±0.2* 3.4±0.3

PE-L 2.2±0.2 4.9±0.3* 7.9±0.3* 8.2±0.2* 5.8±0.3* 3.2±0.3

EA-L 2.2±0.2 5.0±0.3* 8.1±0.5* 8.9±0.2* 6.0±0.1* 3.3±0.3

MT-L 2.2±0.5 4.2±0.2* 7.0±0.5* 6.8±0.3* 4.3±0.3 3.4±0.1

PE-S 2.3±0.2 4.9±0.3* 7.8±0.4* 8.1±0.2* 5.3±0.3 3.3±0.4

CL-S 2.4±0.3 4.0±0.4 6.4±0.3* 6.7±0.1* 4.1±0.4 3.3±0.1

MT-S 2.2±0.4 4.3±0.5 7.0±0.2* 7.2±0.1* 4.8±0.3 3.2±0.4

PE-F 2.2±0.2 4.4±0.4 7.2±0.1* 7.8±0.4* 5.2±0.3 3.3±0.3

MT-F 2.2±0.4 4.3±0.5 7.0±0.2* 7.2±0.1* 4.8±0.3 3.2±0.4


________________________________________________________________________



All the values are expressed as mean ± SEM; n = 6; *P < 0.05 significant compared to

control.




of leaves; MT-L – Methanol extract of leaves

Figure 99: Effect of various extracts of Clerodendrum splendens (50 mg/kg, i.p.) on

thermic stimulus-induced pain (Hot plate test).

0 30 60 90 120 1500

3

6

9

12 Control

Standard

PE-L

EA-L

MT-L

PE-S

CL-S

*

*

*

*

* ** *

Time after treatment (min)

Reacti

on t

ime (

sec)

mean ±

S.E

.M.

MT-S

PE-F

MT-F

*


control.






________________________________________________________________________



6.7.3. Acetic acid-Induced Writhing Test

Procedure:

Peripheral nociceptive activity was evaluated using Acetic acid-Induced Writhing Test

(Koster et al., 1959; Nirmal et al., 2012). Mice were divided into ten groups of six

animals each. The first group served as control and was treated with vehicle only (10%

Tween 80 in distilled water). The second group was administered standard drug

paracetamol (50 mg/kg, i.p.). The animals of third to tenth groups were treated with

petroleum ether extract of leaf, ethyl acetate extract of leaf, methanol extract of leaf,

petroleum ether extract of stem, chloroform extract of stem, methanol extract of stem,

petroleum ether extract of flower and methanol extract of flower of Clerodendrum

splendens (50 mg/kg, i.p., each), respectively, 30 min before intra-peritoneal injection of

0.6% solution of acetic acid (10 ml/kg). All the extracts and standard drug were dissolved

in the vehicle. After acetic acid injection, the mice were observed for the number of

writhing responses for the period of 30 min for each animal.


Ethyl acetate extract (50 mg/kg, i.p.) of leaves of Clerodendrum splendens produced

significant inhibition of writhing reaction induced by acetic acid compared to control

group. Petroleum ether extract of leaves, stem and flowers also showed significant

activity (Table 61 and Figure 100).

The abdominal constriction produced after administration of acetic acid is related to

sensitization of nociceptors to prostaglandins. Chemical and physical stimuli activate the

Ca2+ dependent translocation of group IV cytosolic PLA2 (cPLA2), which has high

affinity to arachidonic acid, to the membrane where it hydrolyzes the sn-2 ester bond of

membrane phospholipids, releasing arachidonate. Arachidonate is metabolized to the

cyclic endoperoxide prostaglandin G (PGG) and H (PGH) by the cyclo-oxygenase (COX)

and hydroperoxidase (HOX) activities of the prostaglandin G/H synthases.

Further isoeicosanoids, a family of eicosanoids isomers, are formed non-enzymatically by

direct free radical based attack on arachidonic acid and related substrates (Lawson et. al.,

1999).

Standard drug Paracetamol, exert their antipyretic action by largely inhibiting

prostaglandin (E-type) protection in the hypothalamus (Rang et.al., 1999). It is, therefore,


________________________________________________________________________



postulated that ethyl acetate extract (50 mg/kg, i.p.) of leaves of Clerodendrum splendens

may probably exert its analgesic effect by either inhibiting the enzymatic synthesis of

prostanoids or by inhibiting the non-enzymatic free radical based synthesis of

isoprostanes.


Acetic acid-Induced Writhing on mice.

Treatment Number of writhing’s

Control 61.17±1.2

Paracetamol (50 mg/kg, i.p.) 28.33±2.4*

PE-L 38.14±1.4*

EA-L 35.1±1.2*

MT-L 52.12±1.7

PE-S 40.2±3.2*

CL-S 57.5±1.8

MT-S 50.12±2.1

PE-F 43.12±1.3*

MT-F 47.1±2.0


control.






________________________________________________________________________




Acetic acid-Induced Writhing on mice.

Control Standard PE-L EA-L MT-L PE-S CL-S MT-S PE-F MT-F

0

25

50

75

Control

Standard

PE-L

EA-L

MT-L

PE-S

CL-S

MT-S

PE-F

MT-FTreatment

Num

ber

of

wri

thin

gs

mean ±

S.E

.M.

*

*

**

*


control.






________________________________________________________________________



6.7.5. Assessment of Anti-inflammatory activity by Carrageenan Induced Rat Paw

Edema

Procedure:

Anti-inflammatory activity was evaluated using carrageenan-induced hind paw edema

method (Winter et al., 1962; Nirmal et al., 2012). Wistar rats of either sex were divided

into ten groups of six animals each. The first group served as control and received only

vehicle (10% Tween 80 in distilled water), and the second group was administered

standard drug ibuprofen (50 mg/kg, i.p.). The animals of the third to tenth groups were

treated with petroleum ether extract of leaf, ethyl acetate extract of leaf, methanol extract

of leaf, petroleum ether extract of stem, chloroform extract of stem, methanol extract of

stem, petroleum ether extract of flower and methanol extract of flower of Clerodendrum

splendens (50 mg/kg, i.p., each), respectively. All the extracts and standard drug were

dissolved in the vehicle. After 30 min of the above treatments, 0.05 ml of 1%w/v

carrageenan in saline was injected into the subplantar tissue of the left hind paw of the

animals. The degree of paw edema of all the groups was measured plethysmometrically

at 0, 30, 60, 90 and 120 min after the administration of carrageenan to each group; 0 min

readings are the initial paw volume of animals.

6.7.6. Results and Discussion

In the acute inflammation model i.e. Carrageenan Induced Rat Paw Edema Method, ethyl

acetate extract of leaf, petroleum ether extract of leaf, stem and flower (50 mg/kg, i.p.)

produced significant (P<0.05) inhibition of paw edema as compared to the control. The

activity of extracts was compared with standard drug ibuprofen (50 mg/kg, i.p.). (Table

62 and Figure 101)

Carrageenan-induced edema is a biphasic response. The first phase is mediated through

the release of histamine, serotonin and kinins, whereas the second phase is related to the

release of prostaglandin and slow reacting substances which peak at 3 h (Vinegar et al.,

1969). In case of analgesia, prostaglandins and bradykinins were suggested to play an

important role in the pain process (Dray and Perkin, 1993). Some sterols and triterpenes

are responsible for anti-inflammatory and analgesic activity (Singh et al., 1997). Ethyl

acetate extract of leaf and Petroleum ether extracts of leaf, stem and flower of


________________________________________________________________________



Clerodendrum splendens showed significant anti-inflammatory activity. Thus these

extracts might be responsible for anti-inflammatory activity.

Airway obstruction/bronchoconstriction or airway hyper-responsiveness in asthma are

believed to be a direct consequence of airway wall inflammation (Holt et al., 1999;

Prasad et al., 2000). This proposed mechanism is consistent with previous findings that

anti-inflammatory plant principles have shown to act through control of adrenocorticoid

hormone and immunosuppression, respectively (Barik et al., 1992; Singh et al., 1997).

Table 62: Effect of various extracts of Clerodendrum splendens on Carrageenan-Induced

Rat Paw Edema.

Treatment

Mean increase in paw volume ml±SEM

Time in min

0 30 60 90 120

Carrageenan

(Control) 0.24±0.01 0.48±0.03 0.78±0.09 0.85±0.12 0.89±0.14

Ibuprofen

(50 mg/kg, i.p.) 0.24±0.03 0.31±0.07* 0.30±0.07* 0.27±0.06* 0.26±0.13*

PE-L 0.25±0.01 0.36±0.05 0.36±0.09* 0.34±0.06* 0.30±0.05*

EA-L 0.24±0.05 0.34±0.05 0.34±0.01* 0.30±0.02* 0.28±0.06*

MT-L 0.25±0.07 0.40±0.09 0.47±0.07 0.49±0.01 0.40±0.09

PE-S 0.24±0.14 0.35±0.09 0.35±0.07* 0.32±0.09* 0.30±0.12*

CL-S 0.25±0.07 0.41±0.02 0.48±0.01 0.51±0.12 0.42±0.13*

MT-S 0.25±0.09 0.36±0.07 0.35±0.09* 0.35±0.07* 0.32±0.11*

PE-F 0.25±0.05 0.35±0.09 0.35±0.01* 0.34±0.07* 0.31±0.06*

MT-F 0.25±0.13 0.37±0.17 0.36±0.09* 0.35±0.02* 0.32±0.04*

All values are expressed as mean±SEM; n=6, * P<0.05 significant compared to control.






________________________________________________________________________



Statistical analysis

All the data is presented as mean ± SEM. Data was analyzed by one-way ANOVA




Carrageenan-Induced Rat Paw Edema.

0 30 60 90 1200.00

0.25

0.50

0.75

1.00Carrageenan

Standard

PE-L

EA-L

MT-L

PE-S

CL-S

***

***

Time in min

Mean i

ncre

ase i

n p

aw

volu

me (

ml±

SE

M)

MT-S

PE-F

MT-F

All values are expressed as mean±SEM; n=6, * P<0.05 significant compared to control.





6. pharmacological screening 6.1. anti-asthmatic activity...

Documents