Pharmacognostic, Phytochemical and Pharmacological studies on 145 Clerodendrum splendens. Family- Verbenaceae.

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1 Anti-asthmatic activity 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 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). Pharmacognostic, Phytochemical and Pharmacological studies on 145

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) Amine mediators 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) 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) Lipid- derived mediators 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, Pharmacognostic, Phytochemical and Pharmacological studies on 146

3 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-A 4 hydrolase in neutrophils, is an extremely potent activator of neutrophils, causing aggregation chemotaxis and degranulation (Peter and White, 1984) 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) Animal required for the pharmacological screening A) Male albino mice (Swiss strain) weighing g were used for anti-cataleptic activity and analgesic activity. B) Colony-bred adult Wistar rats ( g) were used for anti-inflammatory activity. C) Male guinea pigs ( g) were used for evaluating anti-histaminic action of extracts on isolated ileum and Guinea pig (Dunkey-Hartley strain) of either sex weighing ( g) were used for Histamine aerosol induced Bronchoconstriction 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 C and relative humidity %. The cycle of 12-hour light and 12 hour dark was maintained. The experimental protocol was approved by Institutional Animal Ethical Committee 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 Pharmacognostic, Phytochemical and Pharmacological studies on 147

4 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 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) 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. Pharmacognostic, Phytochemical and Pharmacological studies on 148

5 6.5. Assessment of Anti-asthmatic activity 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, NaH2PO 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 Result and Discussion Histamine ( 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 ( 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 Pharmacognostic, Phytochemical and Pharmacological studies on 149

6 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 PE-S CL-S MT-S PE-L EA-L MT-L PE-F MT-F histamine (80 μg/ml) 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. Pharmacognostic, Phytochemical and Pharmacological studies on 150

7 % Inhibition of smooth muscle contraction CHAPTER 6 Figure 92: Effect of various extracts of C. splendens (80 µg/ml) on histamine-induced contraction on guinea pig ileum PE-S CL-S MT-S PE-L EA-L MT-L PE-F MT-F Histamine (µg/ml) 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 Pharmacognostic, Phytochemical and Pharmacological studies on 151

8 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 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 Result and Discussion 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). Pharmacognostic, Phytochemical and Pharmacological studies on 152

9 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 Pharmacognostic, Phytochemical and Pharmacological studies on 153

10 representing its H1 receptor antagonistic activity and support the plants by its antiasthmatic 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 ± ± ± ± 0.91 CHM 1mg/kg ± ± ± ± 1.03 PE-S 50 mg/kg ± ± ± ± 0.80 PE-S 100 mg/kg ± ± ± ± 0.94 CL-S 50 mg/kg ± ± ± ± 0.55 CL-S 100 mg/kg ± ± ± ± 1.07 MT-S 50 mg/kg ± ± ± ± 0.45 MT-S 100 mg/kg ± ± ± ± 0.40 PE-F 50 mg/kg ± ± ± ± 0.72 PE-F 100 mg/kg ± ± ± ± 0.63 MT-F 50 mg/kg ± ± ± ± 0.82 MT-F 100 mg/kg ± ± ± ± 0.92 PE-L 50 mg/kg ± ± ± ± 1.29 PE-L 100 mg/kg ± ± ± ± 1.54 EA-L 50 mg/kg ± ± ± ± 1.33 EA-L 100 mg/kg ± ± ± ± 1.32 MT-L 50 mg/kg ± ± ± ± 0.50 MT-L 100 mg/kg ± ± ± ± 0.43 Pharmacognostic, Phytochemical and Pharmacological studies on 154

11 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 PE-S CL-S MT-S PE-F MT-F PE-L EA-L MT-L 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 PE-S CL-S Pharmacognostic, Phytochemical and Pharmacological studies on 155

12 MT-S PE-F MT-F PE-L EA-L MT-L 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 Pharmacognostic, Phytochemical and Pharmacological studies on 156

13 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 Pharmacognostic, Phytochemical and Pharmacological studies on 157

14 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 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 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 Pharmacognostic, Phytochemical and Pharmacological studies on 158

15 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 haloperidolinduced catalepsy in mice 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 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). Pharmacognostic, Phytochemical and Pharmacological studies on 159

16 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± ± ± ± ± ± ±0.2 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± ±0.2 87±0.3 62±0.3 49±0.2 36±0.3 CL-S 50 25± ± ± ± ± ± ±0.2 MT-S 50 25±0.1 80± ± ±0.3 97±0.2 68±0.2 51±0.2 PE-F 50 21±0.7 72± ±0.4 97±0.4 71±0.4 52±0.5 37±0.1 MT-F 50 22±0.6 85± ± ± ±0.3 98±0.7 52±0.7 PE-L 50 25±0.8 69± ±0.6 92±0.7 68±0.2 51±0.6 37±0.4 EA-L 50 21±0.3 58± ±0.7 78±0.5 67±0.1 48±0.7 29±0.7 MT-L 50 21±0.4 87± ± ± ±0.7 97±0.3 53±0.5 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). 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 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. 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. Pharmacognostic, Phytochemical and Pharmacological studies on 160

17 Figure 97: Effect of various extracts of C. splendens (50 mg/kg, i.p.) on Clonidine- Induced Catalepsy in mice. Duration of catalepsy (sec) Mean ± SEM Time interval (min) Control PHM PE-S CL-S MT-S PE-F MT-F PE-L EA-L MT-L 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). 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 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 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. Pharmacognostic, Phytochemical and Pharmacological studies on 161

18 Result for Haloperidol-Induced Catalepsy None of the extracts of C. splendens inhibited haloperidol induced catalepsy significantly (Table 59 and Figure 98) Table 59: Effect of various extracts of Clerodendrum splendens (50 mg/kg, i.p.) on 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± ± ± ± ± ± ±0.2 PE-S 50 23± ± ± ± ± ± ±0.3 CL-S 50 25± ± ± ± ± ± ±0.2 MT-S 50 24± ± ± ± ± ± ±0.4 PE-F 50 22± ± ± ± ± ± ±0.7 MT-F 50 23± ± ± ± ± ± ±0.1 PE-L 50 19± ± ± ± ± ± ±0.7 EA-L 50 25± ± ± ± ± ± ±0.5 MT-L 50 23± ± ± ± ± ± ±0.6 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). 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. 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. Pharmacognostic, Phytochemical and Pharmacological studies on 162

19 Figure 98: Effect of various extracts of C. splendens (50 mg/kg, i.p.) on Haloperidol- Induced Catalepsy in mice. Duration of catalepsy (sec) Mean ± SEM Time interval (min) Control PE-S CL-S MT-S PE-F MT-F PE-L EA-L MT-L 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). 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 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., Pharmacognostic, Phytochemical and Pharmacological studies on 163

20 (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. Pharmacognostic, Phytochemical and Pharmacological studies on 164

21 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 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 ± 0.2 o C, 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 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- Pharmacognostic, Phytochemical and Pharmacological studies on 165

22 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). Table 60: Effect of various extracts of Clerodendrum splendens (50 mg/kg, i.p.) on thermic stimulus-induced pain (Hot plate test). Treatment Reaction time (Sec) Time after treatment (min) Control 2.3± ± ± ± ± ±0.2 Pentazocine (10 mg/kg, i.p.) 2.8± ± ± ± ± ±0.3 PE-L 2.2± ± ± ± ± ±0.3 EA-L 2.2± ± ± ± ± ±0.3 MT-L 2.2± ± ± ± ± ±0.1 PE-S 2.3± ± ± ± ± ±0.4 CL-S 2.4± ± ± ± ± ±0.1 MT-S 2.2± ± ± ± ± ±0.4 PE-F 2.2± ± ± ± ± ±0.3 MT-F 2.2± ± ± ± ± ±0.4 Pharmacognostic, Phytochemical and Pharmacological studies on 166

23 All the values are expressed as mean ± SEM; n = 6; P < 0.05 significant compared to control. 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 99: Effect of various extracts of Clerodendrum splendens (50 mg/kg, i.p.) on thermic stimulus-induced pain (Hot plate test). Reaction time (sec) mean ± S.E.M. 12 Control Standard 9 PE-L EA-L 6 MT-L PE-S 3 CL-S Time after treatment (min) MT-S PE-F MT-F All the values are expressed as mean ± SEM; n = 6; P < 0.05 significant compared to control. 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. Pharmacognostic, Phytochemical and Pharmacological studies on 167

24 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 Result and Discussion 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, Pharmacognostic, Phytochemical and Pharmacological studies on 168

25 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. Table 61: Effect of various extracts of Clerodendrum splendens (50 mg/kg, i.p.) on 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 All the values are expressed as mean ± SEM; n = 6; P < 0.05 significant compared to control. 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. Pharmacognostic, Phytochemical and Pharmacological studies on 169

26 Figure 100: Effect of various extracts of Clerodendrum splendens (50 mg/kg, i.p.) on Acetic acid-induced Writhing on mice. Number of writhings mean ± S.E.M Control Standard PE-L EA-L MT-L PE-S CL-S MT-S PE-F MT-F Treatment Control Standard PE-L EA-L MT-L PE-S CL-S MT-S PE-F MT-F All the values are expressed as mean ± SEM; n = 6; P < 0.05 significant compared to control. 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. Pharmacognostic, Phytochemical and Pharmacological studies on 170

27 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 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 Pharmacognostic, Phytochemical and Pharmacological studies on 171

28 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 Carrageenan (Control) Ibuprofen (50 mg/kg, i.p.) 0.24± ± ± ± ± ± ± ± ± ±0.13 PE-L 0.25± ± ± ± ±0.05 EA-L 0.24± ± ± ± ±0.06 MT-L 0.25± ± ± ± ±0.09 PE-S 0.24± ± ± ± ±0.12 CL-S 0.25± ± ± ± ±0.13 MT-S 0.25± ± ± ± ±0.11 PE-F 0.25± ± ± ± ±0.06 MT-F 0.25± ± ± ± ±0.04 All values are expressed as mean±sem; n=6, P<0.05 significant compared to control. 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. Pharmacognostic, Phytochemical and Pharmacological studies on 172

29 Statistical analysis All the data is presented as mean ± SEM. 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. Figure 101: Effect of various extracts of Clerodendrum splendens (50 mg/kg, i.p.) on Carrageenan-Induced Rat Paw Edema. Mean increase in paw volume (ml±sem) Time in min Carrageenan Standard PE-L EA-L MT-L PE-S CL-S MT-S PE-F MT-F All values are expressed as mean±sem; n=6, P<0.05 significant compared to control. 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. Pharmacognostic, Phytochemical and Pharmacological studies on 173