Antinociceptive and anti-inflammatory properties of Ruellia tuberosa

Abstract

The ethanol extract of Ruellia tuberosa L. (Acanthaceae) was evaluated for its antinociceptive and anti-inflammatory properties in experimental mice and/or rat models. In the hot-plate test, the group that received a dose of 300 mg/kg showed maximum time needed for the response against thermal stimuli (5.11 ± 0.12), which was similar to that of diclofenac sodium (5.96 ± 0.18), a well-known painkiller. The maximum possible analgesia (MPH) was 1.93 for the extract dose 300 mg/kg, while that for diclofenac was 2.29 after 60 min of administration in the hot tail-flick method. The extract at 500 and 250 mg/kg doses showed significant reduction in acetic acid-induced writhing in mice with a maximum effect of 63.21% reduction at 500 mg/kg dose, which was similar to positive control diclofenac sodium (66.98%). The extract also demonstrated significant inhibition in serotonin and egg albumin-induced hind paw edema in rats at the doses 100, 200 and 300 mg/kg (serotonin-induced edema 35.85, 46.78 and 55.18%; egg albumin-induced edema 42.96, 48.30, and 55.61% inhibition after 1-4 h). The anti-inflammatory properties exhibited by the extract were comparable to that of indomethacin at a dose of 5 mg/kg (serotonin-induced edema 53.22; egg albumin-induced edema 57.01% inhibition after 4 h).

Keywords::

• Ruellia tuberaosa
• Acanthaceae
• antinociceptive
• anti-inflammatory

Introduction

Ruellia tuberosa L. (common name: “patpatey”), one of the approximately 50 species of perennial herbs of the genus Ruellia, belongs to the subfamily Acanthoideae and the family Acanthaceae (Ahmed, 1997; Lans, 2006; GRIN Database, 2007). While this plant is believed to be native to the countries of South America, it is also widely naturalized in a number of countries of other continents (GRIN Database, 2007). In Bangladesh, R. tuberosa grows all over the country, mostly on non-flooded land. This plant is well known for its traditional uses as an antiseptic, depurative, diaphoretic, diuretic, emetic and purgative, and for the treatment of bronchitis, constipation, bladder stone, cystitis, fever, leprosy, gonorrhea and other venereal diseases (Ahamed, 1997; Lans, 2006; Phytochemical and Ethnobotanical Databases, 2007). Ruellia tuberosa was also reported to be used as an abortifacient and to treat uterine fibroids (Balick et al., 2000; Andreana et al., 2002). Roots of this plant have been used for reducing toxicity and healing associated with urinary tract inflammation (Lans, 2006). Previous bioactivity studies on this plant revealed its antioxidant (Chen et al., 2006) and antimicrobial (Wiart et al., 2005) properties; phytochemical analysis led to the isolation of a variety of plant secondary metabolites, including long-chain alkane derivatives (Misra et al., 1997), flavonoids (Wagner et al., 1971; Nair & Subraman, 1974; Behari et al., 1981), sterols and terpenoids (Behari et al., 1981; Singh et al., 2002; Phytochemical and Ethnobotanical Databases, 2007). In continuation of our studies on the bioactivity of Bangladeshi medicinal plants (Rouf et al., 2006; Datta et al., 2007; Saha et al., 2007; Uddin et al., 2005, 2007a, 2007b, 2007c), we now report on the antinociceptive and anti-inflammatory properties of the ethanol extract of Ruellia tuberosa in animal models.

Materials and methods

Plant material

The aerial parts of Ruellia tuberosa L. were collected from Khulna, Bangladesh, in July 2006. The plant was identified by Professor Salar Khan of the Bangladesh National Herbarium and a voucher specimen (MAA06-06) representing this collection has been retained in the herbarium of Pharmacy Discipline, University of Khulna, Khulna, Bangladesh.

Extraction

Dried ground aerial parts (400 g) were extracted with absolute ethanol (IL) using a Soxhlet apparatus. The extract was concentrated by evaporation under reduced pressure at 40°C using a Buchi rotary evaporator to yield a gummy concentrate of greenish extract.

Animals

Wistar rats (200–250 g) and albino mice (20–25 g) of either sex were used in this investigation. Wistar rats and Swiss albino mice were obtained from the animal house of the International Centre for Diarrheal Disease and Research, Bangladesh (ICDDR, B). Animals were maintained under standard environmental conditions and had free access to feed and water. Experiments on animals were performed strictly in accordance with the guidelines provided by the Institutional Animal Ethics Committee. Albino mice (n = 7 per group) were used for antinociceptive activity, and Wistar rats (n = 5 per group) were used for anti-inflammatory screening and divided into five different groups. The first group served as control animals, and they were treated with 1% Tween 80. The animals in the second group were treated with positive control. The next three groups of animals were treated with the alcohol extract at three different doses (100, 200, and 300 mg/kg).

Test samples and positive controls

A suspension of the EtOH extract of R. tuberosa was prepared in Tween 80 (1%) using distilled water. Diclofenac sodium (100 mg/kg) and indomethacin (5 mg/kg) were used as positive controls. All test samples and the positive controls were administered orally by a feeding needle.

Assessment of antinociceptive activity

The hot-plate method

Albino mice were placed in an aluminum hot-plate kept at a temperature of 55 ± 1°C for a maximum time of 10 sec (Franzotti et al., 2000). Reaction time was recorded when animals licked their fore and hind paws, and jumped, following oral administration of the extract (100, 200, and 300 mg/kg) or diclofenac sodium at a dose of 100 mg/kg.

The tail immersion/hot tail-flick method

Mice were treated with 1% Tween 80 (10 mL/kg), diclofenac sodium (100 mg/kg) and three doses of the extract (100, 200, and 300 mg/kg). The antinociceptive effect of the test substances was determined by the hot tail-flick method described by Sewell and Spencer (1976). One to two cm of the tail of mice was immersed in warm water kept constant at 50 ± 1°C. The reaction time was the time taken by the mice to deflect their tails. The first reading is discarded and the reaction time was taken as a mean of the next two readings. The latent period of the tail-flick response was taken as the index of antinociception and was determined before and at 0, 30, and 60 min after the administration of drugs. The maximum reaction time was fixed at 10 sec. The maximum possible analgesia (MPA) was calculated as:

(Test reaction time 2 Saline reaction time) / (10 2 Saline reaction time)

Acetic acid-induced writhing test

The acetic acid-induced writhing test employed in this study was adapted from those previously published in the literature (Koster et al., 1959; Williamson et al., 1996; Zakaria et al., 2001; Silva et al., 2003). Experimental animals were randomly selected and divided into four groups denoted as group I, group II, group III, group IV, consisting of 7 mice in each group. Each group received a particular treatment, i.e., control, positive control and the two doses of the extract. Group I served as the control and received only distilled water and Tween 80. Group II received diclofenac sodium (100 mg/kg), the positive control, for comparison of potencies. The last two groups, i.e., group III and group IV, were treated orally with the crude extract suspensions. A 30-min interval was given to ensure proper absorption of the administered substances. Then each group was treated with intraperitoneally (i.p.) administered 0.2 mL of a 3% acetic acid solution. The number of writhes (i.e., abdominal contractions and stretches) that occurred within the first 20 min following acetic acid administration were counted and recorded. The recorded numbers of acetic acid-induced writhes that occurred with the positive control and test group, i.e., mice treated with the extract, were compared.

Assessment of anti-inflammatory activity

The serotonin-induced rat paw edema assay

Paw edema was induced in the right hind paw by sub-plantar injection of 0.05 mL of 1% freshly prepared solution of serotonin (Mukherjee et al., 1997). The volume of injected paws and contra-lateral paws were measured at 1, 2, 3, and 4 h intervals using a plethysmometer. Extract of Ruellia tuberosa at three different doses (100, 200, and 300 mg/kg) was administered to three groups of animals, and the remaining groups of animals received 1% Tween 80 (negative control 10 mL/kg) and indomethacin (5 mg/kg) for assessing comparative pharmacological significance.

The egg albumin-induced paw edema assay in rats

This test was performed, with suitable modification, according to the method described in the literature (Winter et al., 1962; Ghule et al., 2006). Four groups of male and female Wistar rats were pretreated as follows: group I, 1% aq. Tween 80; groups III, IV and V, 100, 200 and 300 mg/kg, per oral (p.o) of the extracts, respectively, and group II with indomethacine (5 mg/kg, i.p.). After 30 min, each group was injected with 0.5 mL raw egg albumin sub-plantar to the left hind-paw. Edema was assessed in terms of volume of distilled water displaced by the paw before and at 1, 2, 3, and 4 hours after induction of inflammation. The level of inhibition of edema was calculated for each extract using the following equation (Perez, 1996): Inhibition (%) = 100[12 (a/b)] where a = mean paw volume of treated animals after egg albumin injection, b = mean paw volume of control animals after egg albumin injection.

Statistical analysis

Experimental values were expressed as mean ± SEM. Independent sample t-test was carried out for statistical comparison. Statistical significance was considered to be indicated by a p value < 0.05 in all cases.

Results and discussion

Analgesic and anti-inflammatory activity of the EtOH extract of the aerial parts of R. tuberosa was determined by a series of well established and validated experimental methods using mice and/or rat models.

Table 1 shows the results of the hot-plate test. Three doses of extracts of R. tuberosa increased the reaction time in a dose-dependent manner to the thermal stimulus. The highest nociception inhibition of thermal stimulus was exhibited with a dose of 300 mg/kg of extract which had the maximum time needed for the response against thermal stimuli (5.11 ± 0.12). This result was comparable to the response observed with the positive control diclofenac sodium (5.96 ± 0.18).

Table 1.  Effect of Ruellia tuberosa ethanol extract in mice observed in the hot plate test.

Treatment	Dose (mg/kg, p.o.)^*	Response time (sec)
		0 min (Latency)	15 min	30 min	45 min
Control (1% aq. Tween 80 )	10 mL/kg	1.443 ± 0.183	2.485 ± 0.164	2.942 ± 0.292	2.714 ± 0.212
Diclofenac Na	100 (i.p.)	1.885 ± 0.086^*	5.057 ± 0.198^*	5.757 ± 0.303^*	5.957 ± 0.182^*
Ruellia tuberosa extract	100	2.142 ± 0.245^*	4.285 ± 0.162^*	4.342 ± 0.128^*	4.328 ± 0.096^*
	200	2.114 ± 0.193^*	4.471 ± 0.188^*	4.614 ± 0.142^*	4.828 ± 0.357^*
	300	2.128 ± 0.186^*	4.642 ± 0.377^*	5.057 ± 0.127^*	5.114 ± 0.118^*

^*Beginning 30 min after oral administration of test agents (or 15 min after diclofenac Na), the nociceptive response was measured every 15 min over a 45-min period. Each datum represents the latency of nociceptive responses (sec) ± SEM (n = 7) *p <0.05 compared with the control group (Student’s t-test).

Table 2 presents the results of the tail immersion/hot tail-flick test. Three doses of the extract increased the reaction time in a dose-dependent manner to the thermal stimulus. The highest nociception inhibition of thermal stimulus was observed with the dose of 300 mg/kg of the extract (5.07 ± 0.60), which was comparable to the response of diclofenac sodium (5.43 ± 0.34).

Table 2.  Effect of Ruellia tuberosa ethanol extracts in mice observed in the hot tail-flick test.

Treatment	Dose (mg/kg, p.o.)^*	Response time (sec)
		0 min	30 min	60 min
Control (1% aq. Tween 80 )	10 mL/kg	2.571 ± 0.398	2.114 ± 0.183	2.857 ± 0.281
Diclofenac Na	100 (i.p.)	2.728 ± 0.210 (MPA 0.021)	4.085 ± 0.825^** (MPA 1.76)	5.428 ± 0.340^** (MPA 2.285)
Ruellia tuberosa extract	100	2.585 ± 0.326 (MPA 0.002)	3.042 ± 0.263^** (MPA 0.717)	3.757 ± 0.241^** (MPA 0.614)
	200	2.542 ± 0.361 (MPA 0.006)	3.628 ± 0.337^** (MPA 1.302)	4.071 ± 0.395^** (MPA 0.928)
	300	2.628 ± 0.196 (MPA 0.008)	3.814 ± 0.429^** (MPA 1.488)	5.071 ± 0.595^** (MPA 1.928)

^*Beginning 30 min after oral administration of test agents (or 15 min after diclofenac Na), the nociceptive response was measured every 30 min over a 60-min period. Each datum represents the latency of nociceptive responses (sec) ± SEM (n = 7) *p <0.05 compared with the control group (Student’s t-test).

MPA (maximum possible analgesia) = (Test reaction time 2 Saline reaction time)/(10 2 Saline reaction time).

In the acetic acid-induced writhing test (Table 3), a dose-dependent antinociceptive effect was noted with the extract. Maximum percentage of inhibition of writhing response (63.21%) was observed with the dose of 500 mg/kg of the extract. This response was quite similar to that observed with diclofenac sodium (100 mg/kg) causing 66.98% pain inhibition.

Table 3.  Effect of Ruellia tuberosa ethanol extract in mice observed in the acetic acid-induced writhing test.

Treatment	Dose (mg/kg)^*	Route of administration	Writhings^**	% of writhing	% of writhing inhibition
Control (1% aq. Tween 80)	10 mg/kg	p.o.	15.145 ± 1.511	100 ± 3.06	0%
Diclofenac Na	100	i.p.	5.0 ± 0.554^a	33.02 ± 3.66	66.98%
Ruellia tuberosa extract	250	p.o.	7.57 ± 0.945^a	49.99 ± 6.241	50.01%
	500	p.o	5.57 ± 1.035^a	36.79 ± 6.83	63.21%

^*Administered 45 min before 0.2 mL of 3% acetic acid administration (10 mL/kg, i.p.).

^**Counted for 20 min, starting 5 min after acetic acid administration.

^aP <0.05 versus control, Student’s t-test; values are mean ± SE (N = 7).

Table 4 displays the results of the serotonin-induced rat paw edema test. Administration of the extract at different doses produced significant inhibition (p < l 0.05) of edema at the end of 4 h with serotonin administration. However, higher doses of 200 and 300 mg/kg exhibited maximum inhibition of paw edema (46.78 and 55.18%), respectively, and this effect was comparable to that produced by indomethacin (53.22%).

Table 4.  Effect of Ruellia tuberosa ethanol extracts in rats observed in the serotonin-induced edema test.

Treatment	Dose (mg/kg, p.o)^*	Edema volume (μL) (% inhibition)
		0 h	1 h	2 h	3 h	4 h
Control (1% aq. Tween 80 )	10 mL/kg	3.4 ± 0.364	7.3 ± 0.547	8.22 ± 0.542	6.96 ± 0.567	7.14 ± 0.351
Indomethacin	5 (i.p.)	2.68± 0.129	4.4 ± 0.570^** (39.726)	4.8 ± 0.651^** (41.605)	4.28 ± 0.681^** (38.505)	3.34 ± 0.540^** (53.221)
Ruellia tuberosa extract	100	2.7 ± 0.335	5.2 ± 0.821^** (28.767)	5.6 ± 0.570^** (31.873)	4.96 ± 0.343^** (28.735)	4.58 ± 0.772^** (35.854)
	200	2.8 ± 0.183	5.4 ± 0.570^** (26.027)	5.6 ± 1.036^** (31.873)	4.58 ± 0.526^** (34.195)	3.8 ± 0.209^** (46.778)
	300	2.8 ± 0.162	4.92 ± 0.227^** (32.602)	5.4 ± 0.670^** (34.306)	3.84 ± 0.586^** (44.847)	3.2 ± 0.497^** (55.182)

^*The initial hind paw volume of the rat was determined volumetrically. Thirty min after test agent administration (p.o.), 1% serotonin in saline was subcutaneously injected in a volume of 0.05 mL into the right hind paw at time 0 and the paw volume was measured at 1 h intervals for 5 h. Each point represents the mean ± SEM of 5 rats. *p <0.05, compared with the control group (Student’s t-test).

Inhibition (%) = 100(1- [a/b]), where a = mean paw volume of treated animals after egg albumin injection; b = mean paw volume of control animals after egg albumin injection.

The effect of the extract at different doses, and the positive control, on paw edema induced by egg albumin, is shown in Table 5. Paw edema in rats reached its peak after 3 h from the egg albumin administration. The maximum percentage of inhibition of edema (55.61%) was observed with the dose of 300 mg/kg of the extract. This effect was similar to that produced by indomethacin (57.01%).

Table 5.  Effect of Ruellia tuberosa ethanol extracts in rats observed in the egg albumin-induced edema test.

Treatment	^*Dose (mg/kg, p.o.)	Edema volume (μL) (% inhibition)
		1 h	2 h	3 h	4 h
Control (1% aq. Tween 80)	10 mL/kg	5.34 ± 0.643	5.74 ± 0.317	6.56 ± 0.851	7.118 ± 0.456
	5 (i.p.)	3.18 ± 0.341^** (40.449)	3.04 ± 0.222^** (47.038)	3.34 ± 0.168^** (49.085)	3.06 ± 0.201^** (57.010)
Ruellia tuberosa extracts	100	5.08 ± 0.230 (4.868)	4.26 ± 0.499^** (25.783)	4.04 ± 0.175^** (38.414)	4.06 ± 0.168^** (42.961)
	200	4.36 ± 0.627 (18.352)	3.98 ± 0.240^** (30.662)	3.78 ± 0.221^** (42.378)	3.68 ± 0.178^** (48.300)
	300	3.96 ± 0.097^** (25.842)	3.4 ± 0.310^** (40.76)	3.24 ± 0.130^** (50.609)	3.16 ± 0.109^** (55.605)

*The initial hind paw volume of the rat was determined volumetrically. Thirty min after test agent administration (p.o.), raw egg albumin in saline was subcutaneously injected in a volume of 0.5 mL into the right hind paw at time 0 and the paw volume was measured at 1 h intervals for 5 h. Each point represents the mean ± SEM of 5 rats. *p <0.05, compared with the control group (Student’s t-test).

Inhibition (%) = 100(1 − [a/b]) where a = mean paw volume of treated animals after egg albumin injection; b = mean paw volume of control animals after egg albumin injection.

In the hot-plate test and the tail immersion in hot water, the nociceptive reaction towards thermal stimuli in mice is a well-validated model for detection of opiate analgesic as well as several types of analgesic drugs from spinal origin (Sewell & Spencer, 1976; Owoyele et al., 2001; Adzu et al., 2003). Acetic acid is a pain stimulus. Intraperitoneal administration of acetic acid (3%) causes localized inflammation by releasing the free arachidonic acid from tissue phospholipids through the action of phospholipase A₂ and other acyl hydrolases (Koster et al., 1959). There are three major pathways in the synthesis of the eicosanoids from arachidonic acid. All the eicosanoids with ring structures, the prostaglandins, thromboxanes and prostacyclines, are synthesized via the cyclooxygenase pathway. The leucotrienes, HETE (hydroxy eicosatetraenoic acids) and HPETE (hydroperoxy eicosatetraenoic acids) are hydroxylated derivatives of straight-chain fatty acids and are synthesized via the lipooxygenase pathway. The released prostaglandins, mainly prostacyclines (PGI₂) and prostaglandin-E, have been reported to be responsible for pain sensation by exciting the Aδ-fibers. Activity in the A δ-fibers causes a sensation of sharp, localized pain (Rang & Dale, 1993).

Analgesic activity was determined by measuring the writhing effect which was produced by administration of the acetic acid and the inhibition of writhing effect produced by the test samples. Any agent that lowers the writhing number demonstrates analgesia by inhibiting prostaglandin synthesis, a peripheral mechanism of pain inhibition. This hypothesis is in consonance with those authors who postulated that acetic acid-induced writhing test methods are useful techniques for the evaluation of peripherally and centrally acting analgesic drugs (Koster et al., 1959; Williamson et al., 1996; Silva et al., 2003). The extract of R. tuberosa decreased the frequency of acetic acid-induced writhing in mice at higher doses. Diclofenac sodium was used as the positive control, which acts by inhibition of prostaglandin synthesis. It reduces inflammation, swelling, and arthritic pain by inhibiting prostaglandin synthesis and/or production (Skoutakis et al., 1988; Todd & Sorkin, 1988; Small, 1989). The drug also affects polymorphonuclear leukocyte function in vitro, thereby reducing chemotaxis, superoxide toxic radical formation, oxygen-derived free radical generation, and neutral protease production (Freeman et al., 1986; Mahgoub, 2002). Diclofenac has also been reported to suppress inflammation induced by various phlogistic agents in experimental animal models (Menasse et al., 1978; Freeman et al., 1986; Al-Tuwaijri & Mustafa, 1992). Thus, the antinociceptive activity displayed by the EtOH extract of R. tuberosa in the hot-plate, the hot tail-flick and the acetic acid-induced writhing tests, similar to the responses of the positive control diclofenac, indicated that the antinociceptive property of this extract might have been mediated both centrally and peripherally.

Experimental results also revealed that administration of the extract inhibited the edema starting from the first hour and during all phases of inflammation, which is probably inhibition of different aspects and chemical mediators of inflammation. The effect of the extract in the inflammation process induced by serotonin suggested that the extract might have affected the time delayed system in a similar fashion to glucocorticoids (Ahamed et al., 2005).

The egg albumin-induced edema method is another useful tool for describing the systemic inflammation mediated by the release of histamine and prostaglandin (Okoli et al., 2005). These observations suggested that the extract might possess centrally and peripherally mediated analgesic properties and exert its peripheral antinociceptive effects by inhibiting the release, synthesis, and/or production of inflammatory cytokines and mediators, including prostaglandins, histamine, polypeptide kinins, and so on. The reduction of the antinociceptive process obtained within the first hour is probably related to reduction in the release of preformed inflammatory agents, rather than to a reduced synthesis of the inflammatory mediators by inhibition of cyclooxygenases and/or lipoxygenases (and other inflammatory mediators).

The results demonstrated significant antinociceptive and anti-inflammatory properties of the ethanol extract of R. tuberosa, which was comparable to those of the positive controls, and indicated that this plant could be a potential source for the discovery and development of newer analgesic and anti-inflammatory “leads” for drug development.

Acknowledgments

Prof. Dr. Abdul Ghani, Pharmacy Department, Stamford University, Bangladesh, is thanked for valuable suggestions about the study design.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.