MATERIALS AND METHODS

Plant material: The roots of the plant C. erythrocarpos were obtained and authenticated at the Centre for Scientific Research into Plant Medicine (CSRPM), Mampong-Akwapim, Ghana in April, 2007 and a voucher specimen (No. FP/08/024) was kept in the Faculty of Pharmacy Herbarium.

Preparation of extract: Chopped roots were sun-dried for four days and powdered using a hammer mill. The ethanolic extract was obtained by cold maceration of the root powder (5 kg) using 70% (v/v) ethanol over a period of 6 days and the filtrate was concentrated under reduced pressure at a temperature of 60°C in a rotary evaporator to obtain a brown syrupy mass. This syrupy mass was then dried to a dark brown semi-solid using a water bath and kept in a dessicator afterwards. Final yield was 450 g; 9% w/w.

Drugs: Formalin, naloxone hydrochloride and theophylline were purchased from Sigma-Aldrich Inc., St. Louis, MO, USA and morphine hydrochloride was purchased from Phyto-Riker, Accra, Ghana.

Animals: ICR mice (20-30 g) of both sexes were purchased from Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana and housed in the animal facility of the Department of Pharmacology, Kwame Nkrumah University of Science and Technology (KNUST). The animals were housed in groups of six in stainless steel cages (34x47x18 cm³) with soft wood shavings as bedding, fed with normal commercial pellet diet (GAFCO, Tema), given water ad libitum and maintained under laboratory conditions (temperature 24-28°C, relative humidity 60-70% and 12 h light-dark cycle). All procedures and techniques used in these studies were in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (NIH, Department of Health and Human Services publication No. 85-23, revised 1985). All protocols used were approved by the Departmental Ethics Committee.

Phytochemical analysis: The presence of saponins, tannins, alkaloids, triterpenes, flavonoids, glycosides and reducing sugars were tested by simple qualitative methods as described by Trease and Evans (1989) and Sofowora (1993).

Formalin-induced nociception: The mouse paw formalin test used was a slight modification of the method of Dubuisson and Dennis (1977) as described by Woode et al. (2009). Briefly, mice were randomly divided into groups of five. Each animal was assigned to one of twenty Perspex test chambers (15x15x15 cm³) and allowed to acclimatize for 30 min before the start of the experiment. Test drugs were given 30 min for i.p. route and 1 h for oral route before intraplantar injection of 10 μL of 5% formalin solution into the right hind paw of mice. Animals were immediately returned individually into the testing chamber. A mirror was placed at 45° angle beneath the chambers to allow an unobstructed view of the injected hind paws. The behavior of the animal was then captured (60 min) for analysis by a camcorder (Everio™ model GZ-MG1300, JVC, Tokyo, Japan) placed in front of the mirror.

Two sets of experiment were carried out: In the first set, the effect of the extract and morphine alone were tested to determine whether the extract had analgesic effect; mice were grouped as follows:

In the second set, the effect of the extract and morphine in the presence of naloxone and theophylline were tested to determine the possible mechanism of action of the extract; mice were grouped as follows:

All drugs were freshly prepared. Extract was prepared in 2% tragacanth mucilage. Drug solutions and suspensions were prepared such that not more than 1 mL of extract was given orally and not more than 0.5 mL of the standard drug was injected intra peritoneally.

Pain responses were scored for 60 min, starting immediately after formalin injection. A nociceptive index was determined for each 5 min time block by measuring the duration of biting/licking of the injected paw (Hayashida et al., 2003). Behavioral responses were scored from the videotapes with the aid of the public domain software JWatcher™ Version 1.0 (University of California, Los Angeles, USA and Macquarie University, Sydney, Australia available at http://www.jwatcher.ucla.edu/). Average nociceptive score for each 5 min time block was calculated by multiplying the frequency and time spent in biting/licking the injected paw. Data were expressed as the Mean±SEM scores between 0-10 and 10-60 min after formalin injection.

Toxicity test: Acute and sub acute toxicity tests were done by administration of high doses of the extract (0.3-3 g kg^-1 p.o.) followed by close monitoring for signs of toxicity for 24 h and 14 days, respectively. Hematological and biochemical parameters were also monitored.

Data analysis: The time-course curves were subjected to two-way (treatmentxtime) repeated measures Analysis of Variance (ANOVA) with Bonferroni’s post hoc test. Nociceptive score for each treatment was calculated in arbitrary unit as the area under the curve (AUC). To determine the percentage inhibition for each treatment, the following equation was used.

Differences in AUCs were analyzed by ANOVA followed by Newman-Keuls’ post hoc test. The (ED₅₀) which is the dose for 50% of the maximal effect was determined for each drug by using an iterative computer least squares method, with the following nonlinear regression (three-parameter logistic) equation:

where, X is the logarithm of dose and Y is the response. Y starts at a (the bottom) and goes to b (the top) with a sigmoid shape.

The fitted midpoints (ED₅₀) of the curves were compared statistically using F-test (Motulsky and Christopoulos, 2003; Miller, 2003). GraphPad Prism for Windows version 4.03 (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses and ED₅₀ determinations. The p<0.05 was considered statistically significant.

RESULTS

Phytochemical analysis: Phytochemical screening revealed the presence of high amount of alkaloids and flavonoids with traces of triterpenes in the crude extract.

Effect of drugs on nociception: Formalin induced a clear nociceptive response exhibited as biting or licking of the injected paw. The nociceptive response was biphasic as previously described by Bars et al. (2001), Saddi and Abbott (2000) and Wang et al. (1999) consisting of an initial intense response to pain beginning immediately after formalin injection and rapidly decaying within 10 min after formalin injection (first phase). This was then followed by a slowly rising but longer-lasting response (second phase) from10-60 min after formalin injection with maximum effect at approximately 20-30 min after formalin injection (Wang et al., 1999). Pain scores were significantly (p<0.05) lower in the drug-treated groups than in the control (Fig. 1).

The CEE (10-300 mg kg^-1 p.o.) dose-dependently decreased formalin-induced nociceptive behavior in both phases. The highest dose of CEE 300 mg kg^-1 had significant effect in phase 1 (F_{3, 16} = 15.25, p<0.001) and phase 2 (F_3,16 = 35.34, p<0.001) giving the greatest inhibition in both the neurogenic (phase 1) 52.78±8.12% and inflammatory phase (phase 2) 86.52±3.79% (Fig. 1a, b). Morphine (1-10 mg kg^-1), an opioid agonist significantly inhibited the formalin-induced nociceptive behavior in both phases in a dose-dependent fashion. The highest dose 10 mg kg^-1 had significant effect in phase 1 (F_3,16 = 9.54, p<0.001) and phase 2 (F_3,16 = 32.05, p<0.001) and produced the greatest inhibition in both the neurogenic (70.28±11.62%) and inflammatory phase (94.80±4.22%) (Fig. 1c, d).

Administration of naloxone and theophylline alone did not produce any significant antinociceptive effect (Fig. 2a, b). The antinociceptive effect of extract in the presence of naloxone and theophylline is shown in Fig. 3a-d. Capparis erythrocarpos (100 mg kg^-1) significantly reduced formalin-induced nociception with a percentage inhibition of 47.54±5.65% (F_3,16 = 15.25 p<0.001) in the first phase and 80.01±3.77% (F_3,16 = 35.34 p<0.001) in the second phase. This antinociceptive effect of the extract was not antagonized by naloxone in both the neurogenic phase (43.44±4.15%) and the inflammatory phase (82.41±0.90%); however, theophylline completely inhibited the first phase and significantly inhibited the second phase with a percentage inhibition of 57.08±1.88%. In the formalin test, naloxone inhibited the antinociceptive effect of morphine in both the neurogenic and inflammatory phases with percentage inhibitions of 9.14±9.14 and 22.00±4.05%, respectively. Theophylline on the other hand, could not inhibit the antinociceptive effect of morphine in both phase 1 and phase 2.

Fig. 1:	Effect of CEE (10-300 mg kg-1 p.o.) and morphine (1-10 mg kg-1 i.p.) (a, c) on the time course of formalin-induced pain in mice. Nociceptive/pain scores are shown in 5 min blocks up to 60 min post formalin injection; the AUC (total response) for phase 1 and phase 2 (b, d). Each column in b and d represent the Mean±SEM. n = 5 **p<0.001; *p<0.05

Fig. 2:	Effect of intra peritonial injection of naloxone and theophyline on the time course (a) and the AUC (total response) (b) of formalin-induced pain in mice

From the ED₅₀ values calculated from the dose response curves in Fig. 4a and b, the extract was found to be approximately 24.6 and 21.98 times less potent than morphine in the first and second phases of the formalin test, respectively.

No remarkable signs of toxicity were observed either immediately or during the post-treatment period even at the highest dose of 3.0 g kg^-1. There were also no changes noted in behavior, activity, posture, or external appearance that were considered to be test drug related and all the animals survived throughout the 24 h and 14 day study period, respectively. There were generally no statistically significant changes in the hematological and biochemical parameters taken.

Fig. 3:	Effect of intra peritonial injection of naloxone and theophyline on the antinociceptive effect of CEE (100 mg kg-1 p.o.) and morphine (3 mg kg-1 i.p.) (a, c) on the time course of formalin-induced pain in mice and the AUC (total response) for phase 1 and phase 2 (b, d). Each column represents the Mean±SEM. (n = 5) ***p<0.0001; **p<0.001; *p<0.05; ns: Not significant

Fig. 4:	Dose response curves of CEE (a) and morphine (b) on the total nociceptive score for the first phase and the second phase of the formalin test in mice. Each point represents Mean±SEM (n = 5)

DISCUSSION

The results of this study show that the ethanolic extract of Capparis erythrocarpos roots has a potent antinociceptive effect against the chemical stimuli provoked by the intraplantar injection of formalin. The formalin test is one of the most frequently used models to explain pain and analgesic mechanisms. Results from the formalin test are usually better than those using mechanical or thermal stimulus (Tjolsen et al., 1992).

Intraplantar injection of formalin evoked a characteristic biphasic licking response. There was an early phase (0-10 min post formalin injection) due to the direct stimulation of nociceptors by formalin that corresponds to acute neurogenic pain with release of substance P and is sensitive to central analgesics (Bars et al., 2001; Szolcsanyi et al., 2004). The late phase involves inflammatory components with the release of different pain mediating substances such as serotonin, histamine, bradikynin and prostaglandins and is sensitive to NSAIDs (Yashpal and Coderre, 1998; Malmberg and Yaksh, 1992), corticosteroids as well as analgesics with central effects (Vasconcelos et al., 2003).

Response to pain (defined by pain scores) was significantly lower in the drug treated groups than the control treated with only formalin. Capparis erythrocarpos extract exhibited analgesic activity in the rat formalin test by significantly lowering the response of the animals to pain in both phases; it however, inhibited the second phase of the formalin response more than the first phase suggesting that C. erythrocarpos could be useful in inflammatory pain. Since the extract had effect on inflammatory pain, it is possible that it has inhibitory effect on inflammatory mediators notably prostaglandins as well as receptor blockade. Furthermore, the potent analgesic activity of the extract in both phases of the formalin test presupposes a possible central analgesic effect in addition to peripheral analgesic effect.

Morphine, an opioid agonist was antinociceptive and inhibited both phases of the formalin response clearly depicting that it is a centrally acting analgesic drug (Bohn et al., 2002; Catheline et al., 1996). The role of opioid receptors located in the Central Nervous System (CNS) and peripheral nervous system in inflammatory pain is well established (Whiteside et al., 2005).

The analgesic effect was further investigated to elucidate the possible mode or mechanism of action(s) of the extract by using the non-selective opioid receptor antagonist naloxone (Herrero and Headley, 1996) and the non-selective adenosine receptor antagonist theophylline (Sawynok et al., 1991). Compounds that mimic or modulate the antinociceptive action of adenosine presents a potential approach to the treatment of pain (Kowaluk et al., 2000; Jarvis et al., 2002).

As expected naloxone reversed the effect of morphine however, naloxone did not reverse the antinociceptive effect of the extract in both phases, suggesting that opioid receptor activation was not involved in the antinociceptive effect of the extract. Antinociceptive effect of C. erythrocarpos was completely reversed in the first phase and significantly reversed in the second phase by theophyline, which suggest the involvement of adenosine in the antinociceptive effect of C. erythrocarpos and more specifically the receptor subtype A₁ which are involved in antinociception could be implicated (Poon and Sawynok, 1999; Whiteside et al., 2005). Adenosine interacts with at least four P1 receptor subtypes coupled with G protein, namely A₁, A_2a, A_2b and A₃ (Fredholm et al., 2001). These receptors can affect nociceptive, inflammatory and neuropathic pain states at peripheral, spinal and supraspinal sites (Sawynok, 1998; Dickenson et al., 2000; Sawynok and Liu, 2003). Current evidence shows that A₁ receptor activation produces antinociception while activation of A_2a and A₃ mediates pronociceptive actions in rodents (Sawynok, 1998; Sawynok et al., 1998). However, detailed research involving the use of specific agonists and antagonists of A₁ receptors are necessary to confirm this assertion. In addition its mechanism of action may involve other pathways which need to be investigated.

The presence of flavonoids, alkaloids and triterpenes in the ethanolic root extract of Capparis erythrocarpos may be responsible for some of its pharmacological properties. As has been reported by several researchers, the presence of many biologically active phytochemicals such as triterpenes, flavonoids, alkaloids, steroids, tannins and glycosides in various plant extracts may be responsible for their respective pharmacological properties (Singh et al., 2002; Narendhirakannan et al., 2007; Agarwal and Rangari, 2003; Mbagwu et al., 2007; Liu et al., 1996). In addition to this, flavonoids are known to target prostaglandins which are involved in the late phase of acute inflammation and pain perception (Rajnarayanan et al., 2001). Further studies are required to establish the specific chemical constituents responsible for the pharmacological actions. However, the antinociceptive effect of the ethanolic root extract of Capparis erythrocarpos was demonstrated by its ability to dose-dependently reduced pain scores in both phases of formalin-induced nociception and since the principal targets of effective pain control are to ameliorate nociception and to reduce threshold of pain sensation this validates the traditional use of the plant in the treatment of conditions associated with pain.

In the toxicity study, using high concentrations of the extract (0.3-3 g kg^-1 p.o.) there were no significant differences found in most of the haematological indices and serum biochemical parameters suggesting that the extract has a high safety profile.

CONCLUSION

The ethanolic extract of Capparis erythrocarpos roots exhibits central analgesic effect with possible involvement of adenosinergic mechanism. This finding provides some scientific support on the traditional use of roots of Capparis erythrocarpos for controlling inflammatory pain conditions.

ACKNOWLEDGMENT

The authors are grateful for the technical assistance offered by Mr. Thomas Ansah and Mr. George Ofei of the Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, KNUST, Kumasi.