Anti-inflammatory, Antinociceptive, and Neuropharmacological Activities of Clerodendron viscosum.

Abstract

The crude methanol extract of Clerodendron viscosum. Vent. (Verbenaceae) leaves was evaluated for its anti-inflammatory, antinociceptive, and neuropharmacological activities. When given orally to rats at doses of 200 and 400 mg/kg of body weight, the extract showed a significant (p < 0.001) anti-inflammatory activity against carrageenan-induced rat paw edema comparable with the standard drug phenylbutazone at the dose of 100 mg/kg of body weight. It also produced a significant writhing inhibition in acetic acid–induced writhing in mice at the oral dose of 250 and 500 mg/kg of body weight (p < 0.001), which was comparable with the standard drug diclofenac sodium at the dose of 25 mg/kg of body weight. Moreover, when given intraperitoneally to albino mice, it potentiated the pentobarbital-induced sleeping time (p < 0.001), decreased the open field score in open field test (p < 0.001), decreased the number of holes crossed from one chamber to the other in the hole-cross test (p < 0.001), and decreased the head dip responses in the hole-board test (p < 0.001) at the dose of 250 and 500 mg/kg of body weight. The overall results tend to suggest the anti-inflammatory, antinociceptive, and central nervous system depressant activities of the crude methanol extract of Clerodendron viscosum..

Keywords:

• Anti-inflammatory activity
• antinociceptive activity
• Clerodendron viscosum
• neuropharmacological activity

Introduction

Clerodendron viscosum. Vent. (Verbenaceae) (synonym Clerodendron infortunatum. Linn.), locally known as “Bhant” or “ghentu”, is a shrub widely distributed throughout Bangladesh, India, Ceylon, and Malaysia. The plant is used as a tonic, aphrodisiac, antipyretic, analgesic, anthelmintic, and for the treatment of blood diseases (Kirtikar & Basu, 1987). The leaf juice is used as a strong anthelmintic, emetic, and mild laxative. Leaves are used as an antiperiodic in malaria and in the treatment of chest complaints with coughs and asthma. It is externally used for tumors, skin diseases, snakebite, and scorpion sting. The roots are also used as an anthelmintic (Ghani, 1998). From the existing information, it is evident that the plant may possess some important biological activities. No report on its biological activity has been found in the literature. The main objective of this study was to evaluate the anti-inflammatory, antinociceptive, and neuropharmacological activities of the methanol extract of Clerodendron viscosum. (C. viscosum.) leaves.

Materials and Methods

Plant material

Leaves of C. viscosum. were collected in August 2003 from the district of Magura, Bangladesh, and were authenticated by the experts at the National Herbarium (accession number: 29762). About 400 g of leaves were dried and powdered with the help of a suitable grinder, 360 g of which was extracted five times, using 200 mL 90% methanol in a Soxhlet apparatus. The extract was evaporated to dryness by rotary evaporator to get a viscous mass. The viscous mass was then kept at room temperature under a ceiling fan, and then preserved in a vacuum desiccator to get a dried extract of 54.25 g (yield: about 15%). This extract was used for pharmacological screening.

Animals

Swiss albino mice of either sex, weighing 20–25 g, bred in the animal house of the Department of Pharmacy, Jahangirnagar University, were used for antinociceptive and neuropharmacological activity studies. On the other hand, Wistar rats of either sex, weighing 180–200 g, purchased from the Animal Research Branch of the International Center for Diarrhoeal Disease and Research, Bangladesh (ICDDR, B), were used for anti-inflammatory activity study. The animals were provided with standard laboratory food and tap water ad libitum. and maintained at natural day/night cycle. All the experiments were conducted in an isolated and noiseless condition.

Chemical group test

The crude methanol extract of leaves of Clerodendron viscosum. was tested for its different chemical groups such as alkaloids, flavonoids, gums, reducing sugars, saponins, steroids, amino acids, and tannins. In each test, 10% (w/v) solution of the extract in 90% ethanol was used unless otherwise mentioned in an individual test (Evans, 1989; Ghani, 1998).

Tests for reducing sugar

Benedict's test: 0.5 mL of the extract was placed in a test tube and then 5 mL Benedict's solution was added to it, boiled for 5 min, and allowed to cool spontaneously.

Fehling's test (standard test): 2 mL of the extract was added in 1 mL of a mixture of equal volumes of Fehling's solutions A and B and was boiled for a few minutes.

Combined reducing sugar test: 1 mL of the extract was boiled with 2 mL of diluted hydrochloric acid for 5 min. After cooling, the mixture was neutralized with sodium hydroxide solution, and then Fehling's test was performed as described above.

Tests for tannins

Ferric chloride test: The extract (5 mL) was placed in a test tube and then 1 mL of 5% ferric chloride solution was added to it.

Potassium dichromate test: The extract (5 mL) was placed in a test tube and then 1 mL of 10% potassium dichromate solution was added.

Test for flavonoids

A few drops of concentrated hydrochloric acid were added to 5 mL of the extract.

Test for saponins

The extract (1 mL) was placed in a graduated cylinder and was diluted to 20 mL with distilled water and shaken gently for 15 min.

Test for gums

The extract (5 mL) was placed in a test tube and then Molish's reagent and sulfuric acid were added to it.

Tests for steroids

Libermann-Burchard test: The extract (1 mL) was placed in a test tube and then 2 mL Libermann-Burchard reagent was added to it.

Sulfuric acid test: The extract (1 mL) was placed in a test tube and 1 mL sulfuric acid was added to it.

Tests for alkaloids

Mayer's test: The extract (2 mL) and dilute hydrochloric acid (0.2 mL) were placed in a test tube and 1 mL of Mayer's reagent was added to it.

Dragendorff's test: The extract (2 mL) and dilute hydrochloric acid (0.2 mL) were placed in a test tube and then 1 mL Dragendorff's reagent was added.

Wagner's test: The extract (2 mL) and dilute hydrochloric acid (0.2 mL) were placed in a test tube then 1 mL of iodine solution (Wagner's reagent) was added.

Hager's test: The extract (2 mL) and dilute hydrochloric acid (0.2 mL) were placed in a test tube then 1 mL of picric acid solution (Hager's reagent) was added.

Anti-inflammatory activity

Anti-inflammatory activity of C. viscosum. was tested by using the carrageenan-induced rat paw edema model (Winter et al., 1962; Ahmed et al., 2004). Rats were randomly divided into four groups, each consisting of six animals. Group I was kept as control giving 1% (v/v) Tween-80 solution in water at a dose of 10 mL/kg of body weight; group II was kept as “positive control” and was given the standard drug phenylbutazone at a dose of 100 mg/kg of body weight; groups III and IV were test groups, treated with extracts at doses of 200 and 400 mg/kg of body weight, respectively. Control vehicle, standard drug, and the extracts were given orally 1 h prior to the injection of 0.1 mL of 1% freshly prepared suspension of carrageenan. The paw volume was measured by using a plethysmometer just before and 1, 2, 3, 4, and 5 h after the carrageenan injection.

Antinociceptive activity

Antinociceptive activity of the methanol extract of C. viscosum. was tested using the model of acetic acid–induced writhing in mice (Whittle, 1964; Ahmed et al., 2004). The experimental animals were randomly divided into four groups, each consisting of 10 animals. Group I was treated as “control” and received 1% (v/v) Tween-80 in water at a dose of 10 mL/kg of body weight; group II was treated as “positive control” and was given the standard drug diclofenac sodium at a dose of 25 mg/kg of body weight; group III and group IV were test groups and were treated with methanol extracts of C. viscosum. at a dose of 250 and 500 mg/kg of body weight, respectively. Control vehicle, standard drug, and the methanol extracts were administered orally 30 min prior to the intraperitoneal injection of 0.7% acetic acid, then after an interval of 15 min; the number of writhings (squirms) was counted for 5 min.

Neuropharmacological activity

Pentobarbital-induced hypnosis

Pentobarbital-induced hypnosis test was carried out by the method of Williamson et al. (1996). The test animals were divided into three groups consisting of seven mice in each group. Group I was the control group, and groups II and III were the experimental groups. The experimental groups were administered the methanol extract of C. viscosum. at doses of 250 and 500 mg/kg body weight intraperitoneally (i.p.), whereas the animals of group I (control) were supplied with distilled water containing 0.1% (v/v) Tween-80 (i.p.) at the dose of 10 mL/kg of body weight. The total sleeping time was recorded for both control and treated groups.

Exploratory behavior

This experiment was performed by (i) open field test (Gupta et al., 1971), (ii) hole-cross test (Takagi et al., 1971), and (iii) hole-board test (Nakama et al., 1972). The test animals were divided into three groups consisting of seven mice in each group. Group I was the control group, and groups II and III were the experimental groups. The experimental groups were administered the methanol extract of C. viscosum. (prepared by distilled water and Tween-80) at doses of 250 and 500 mg/kg of body weight intraperitoneally, whereas the animals of group I (control) were supplied with 0.1% (v/v) Tween-80 (i.p.) at the dose of 10 mL/kg of body weight. The observations were made on 0 min before injection and 30, 60, 120, and 240 min after injections of the test samples and control.

Statistical analysis

Student's t.-test was used to determine a significant difference between the control group and the experimental groups.

Results

Chemical group test

Results of different chemical tests on the methanol extract of C. viscosum. showed the presence of alkaloids, steroids, flavonoids, reducing sugars, saponins, gums, and tannins are shown in Table 1.

Table 1.. Results of chemical group tests.

Chemical group	Test solution	Observation	Inference
Alkaloids	0.1 mL Mayer's reagent	Yellowish buff–colored precipitate was obtained	Alkaloids were present
	0.1 mL Dragendorff's reagent	Orange-brown precipitate was observed
	0.1 mL iodine solution (Wagner's reagent)	Reddish-brown precipitate was obtained
	0.1 mL picric acid solution (Hager's reagent)	Yellowish precipitate was obtained
Steroids	2 mL Libermann-Burchard reagent	Reddish-purple color was obtained
	1 mL sulfuric acid	Chloroform layer acquired reddish-brown color and acid layer showed green fluorescence	Steroids were present
Flavonoids	(a) 1 mL dilute ammonia solution	Greenish-yellow color was obtained	Flavonoids were present
	(b) 1 mL dilute sodium carbonate solution	Pale-yellow color was obtained
	(c) 1 mL dilute sodium hydroxide solution	Yellow color was obtained
Saponins	Shaken in a graduated cylinder for 15 min	One centimeter layer of foam	Saponins were present
Reducing sugars	5 mL Fehling's solution	Brick red–colored precipitate	Reducing sugars were present
	5 mL Benedict's reagent	Brick red–colored precipitate
	2 drops of 5% α-naphthol solution and 1 mL sulfuric acid	Violet-colored ring was formed at the junction of two liquids
Tannins	1 mL 5% ferric chloride solution	Greenish-black precipitate	Tannins were present
	1 ml 10% lead acetate solution	Yellow precipitate
	1 mL 10% potassium dichromate solution	Yellowish-brown precipitate
	1 mL aqueous bromine solution	Brown precipitate
Gums	Molish reagent and sulfuric acid	Red-violet ring was produced at the junction of two liquids	Gums were present
Amino acids	Ninhydrin; pH adjusted between 4 and 8	Purple color was not obtained	Amino acids were absent

Anti-inflammatory activity

The experimental findings from the carrageenan-induced rat paw edema model showed that the methanol extract of C. viscosum. reduced the paw volume significantly from 1 to 5 h. The extract showed highest effects at the third hour where the inhibition was about 29% and 41% at doses of 200 and 400 mg/kg, respectively (p < 0.01; p < 0.001), which were comparable with the standard drug phenylbutazone, where the inhibition was about 51% at the dose of 100 mg/kg of body weight (p < 0.001) (Table 2).

Table 2.. Effect of methanol extract of C. viscosum. on carrageenan-induced rat paw edema.

	Time after carrageenan injection
	1 h	2 h	3 h	4 h	5 h
Animal group/treatment	Increase in paw edema volume (mL) × 1000 ± SEM
Control (1% Tween-80) 10 mL/kg, p.o.	137.00 ± 0.70	164.50 ± 1.02	244.50 ± 0.85	272.00 ± 0.65	231.00 ± 0.97
Positive control Phenylbutazone 100 mg/kg, p.o.	96.30 ± 0.78**	100.50 ± 0.99***	119.20 ± 1.25***	183.00 ± 1.06**	146.75 ± 0.88**
Test group 1 Methanol extract 200 mg/kg, p.o.	114.20 ± 1.25	128.70 ± 1.57	174.40 ± 0.88**	222.00 ± 0.68	183.80 ± 0.86*
Test group 2 Methanol extract 400 mg/kg, p.o.	100.40 ± 1.06	113.30 ± 0.89**	144.30 ± 0.97***	200.80 ± 1.65**	163.60 ± 1.05**

Values are expressed as mean ± SEM (n = 6); *p < 0.05; **p < 0.01; ***p < 0.001 vs. control.

Antinociceptive activity

Table 3 shows the effect of C. viscosum. on acetic acid–induced writhing model in mice. The extract produced about 49% and 62% writhing inhibition at doses of 250 and 500 mg/kg body weight, respectively (p < 0.001), which were comparable with the standard drug diclofenac sodium where the inhibition was about 65% at the dose of 25 mg/kg body weight (p < 0.001).

Table 3.. Effect of methanol extract of C. viscosum. on acetic acid–induced writhing in mice.

Animal Group/treatment	Number of writhes (% writhing)	Inhibition (%)
Control 1% Tween-80 in water 10 mL/kg, p.o.	16.40 ± 0.75 (100)	—
Positive control Diclofenac sodium 25 mg/kg, p.o.	5.80 ± 1.01* (35.36)	64.64
Test group 1 Methanol extract 250 mg/kg, p.o.	8.30 ± 0.65* (50.60)	49.40
Test group 2 Methanol extract 500 mg/kg, p.o.	6.30 ± 0.63* (38.41)	61.59

Neuropharmacological activity

Pentobarbital-induced hypnosis test

Table 4 shows the effect of C. viscosum. on pentobarbital-induced hypnosis in mice. The extract potentiated the pentobarbital-induced sleeping time in mice. The total sleeping time was about 37 and 67 min at doses of 250 and 500 mg/kg of body weight, respectively (p < 0.001), whereas in the control group it was about 20 min.

Table 4.. Effect of C. viscosum. on pentobarbital-induced hypnosis.

Animal group	Treatment	Total sleeping time (min)
I (Control)	0.1% Tween-80 solution 10 mL/kg	19.90 ± 2.01
II (Test group 1)	Methanol extract 250 mg/kg	37.00 ± 2.39
III (Test group 2)	Methanol extract 500 mg/kg	67.50 ± 7.93

Values are expressed as mean ± SEM (n = 7); *p < 0.001 vs. control.

Exploratory behavior test

Test for exploratory behavior in mice was performed by (i) open field test, (ii) hole-cross test, and (iii) hole-board test. It was observed that the extract decreased the number of open field score (p < 0.001), caused decrease in the number of holes crossed from one chamber to another chamber (p < 0.001), and also decreased head dip responses (p < 0.001) in mice at doses of 250 and 500 mg/kg of body weight from 30 to 240 min (Table 5).

Table 5.. Effect of C. viscosum. on exploratory behavior in mice.

	Response at
Group	0 min	30 min	60 min	120 min	240 min
Effect on open field test
I (Control)	86.28 ± 2.11	93.71 ± 1.82	97.57 ± 1.92	104.14 ± 1.42	107.85 ± 1.48
II (Methanol extract) 250 mg/kg	85.85 ± 1.68	79.57 ± 1.25	72.28 ± 1.44	64.57 ± 1.88	61.71 ± 3.32
III (Methanol extract) 500 mg/kg	85.85 ± 2.02	70 ± 2.59	57 ± 2.99	46.42 ± 3.06	42.57 ± 3.48
Effect on hole-cross test
I (Control)	8.42 ± 0.65	9.85 ± 0.46	10 ± 0.49	9.28 ± 0.68	8.42 ± 0.65
II (Methanol extract) 250 mg/kg	7.28 ± 0.68	6.14 ± 0.40	5.0 ± 0.44	3.42 ± 0.37	2.0 ± 0.31
III (Methanol extract) 500 mg/kg	8.43 ± 0.64	6.0 ± 0.49	3.86 ± 0.26	2.14 ± 0.26	1.28 ± 0.18
Effect on hole-board test (head dipping)
I (Control)	14.42 ± 0.65	20.14 ± 1.10	21.57 ± 1.06	19.85 ± 0.59	15.0 ± 0.81
II (Methanol extract) 250 mg/kg	14.0 ± 0.53	12.28 ± 0.36	10.42 ± 0.52	8.425 ± 0.42	5.0 ± 0.41
III (Methanol extract) 500 mg/kg	14.28 ± 0.81	10.85 ± 0.59	8.14 ± 0.74	5.28 ± 0.68	2.85 ± 0.51

Values are expressed as mean ± SEM (n = 7); *p < 0.001 vs. control.

Discussion

The most widely used primary test for the screening of new anti-inflammatory agents is the carrageenan-induced rat paw edema model (Winter et al., 1962). The edema formation is a biphasic event. The initial phase, observed during the first hour, is attributed to the release of histamine, serotonin, and bradykinin (Vinegar et al., 1969; Corea et al., 2005) and the delayed edema is due to the release of bradykinin and prostaglandins and NO (Di Rosa et al., 1971; Flower et al., 1985; Corea et al., 2005). It has been reported that the second phase of edema is sensitive to steroidal and nonsteroidal anti-inflammatory agents (Di Rosa et al., 1971). The extract reduced the paw volume significantly from 1 to 5 h in which the highest effects were found at the third hour. These results tend to suggest the anti-inflammatory activity of the extract at both phases through the suppression of mediator release.

Antinociceptive activity of the extract was tested by the acetic acid–induced writhing model in mice. Acetic acid, which is used to induce writhing, causes algesia by liberation of endogenous mediators like cytokines and eicosanoids with an increase in peritoneal fluid levels of prostaglandin E₂, which then excite the pain-sensitive nerve endings (Whittle, 1964). The extract produced significant writhing inhibition comparable with standard drug diclofenac sodium. Based on this, it could be concluded that it possesses antinociceptive activity. Antinociceptive activity of C. viscosum. may be related with the reduction of the liberation of those inflammatory mediators or by direct blocking of the receptors resulting in peripheral antinociceptive effect. Another possibility could be blockage in the eicosanoid system.

Many centrally depressant agents exert their effect by interfering with the function of the cerebral cortex. A practical method to investigate the probable cortical manifestations of a drug is to check its effect on pentobarbital narcosis as pentobarbital has multifarious effects on the cerebral cortex (Bowman & Rand, 1980). A test substance with CNS-depressant activity can prolong the duration of sleep. The experimental data presented in Table 4 shows that the extract of C. viscosum. significantly increased the duration of the pentobarbital sleeping time. Prolongation of pentobarbital hypnosis is believed to be a sedative and/or hypnotic property (Fujimori, 1965) and could be attributed to inhibition of pentobarbital metabolism (Kaul & Kulkarni, 1978) or a central mechanism involved in the regulation of sleep (N'Gouemo et al., 1994). Moreover, the test for exploratory behavior in mice showed that the extract suppressed the open field score, hole-crossing ability, and head dip responses in mice, which further support the central nervous system depressant activity of the extract.

In conclusion, it could be suggested that the crude methanol extract of C. viscosum. possesses anti-inflammatory, antinociceptive, and central nervous system depressant activities. However, further studies comprising thorough phytochemical investigations of the plant to identify the active principles and confirmation of these activities using other scientific models are essential.