PHYTOTHERAPY RESEARCH, VOL.

11, 211215 (1997)

Analgesic and Antiinammatory Activities of Justicia pectoralis Jacq and its Main Constituents: Coumarin and Umbelliferone
C. S. Lino, M. L. Taveira, G. S. B. Viana and F. J. A. Matos
Department of Physiology and Pharmacology and Laboratory of Natural Products, Federal University of Cear a, Fortaleza, Brazil

The hydroalcohol extract of J. pectoralis (EHA) and its main constituents, coumarin (CM) and umbelliferone (UMB), were studied for possible analgesic and antioedema activities on acetic acid induced writhing in mice and on the carrageenan and dextran paw oedema in rats. The EHA, CM and UMB demonstrated antinociceptive effects, and pretreatment with naloxone did not reverse the antinociception, indicating that the opioid system is not involved. On the other hand, pretreatment with L-arginine reversed the antinociception caused by UMB, suggesting the involvement of the nitric oxide system. The EHA, CM and UMB presented a signicant antioedema effect in the carrageenan model but only CM decreased the rat paw volume in the dextran model, and this effect was not observed with EHA or UMB. 1997 by John Wiley & Sons, Ltd. Phytother. Res. 11, 211215, 1997
(No. of Figures: 1. No. of Tables: 4. No. of Refs: 32.)

Keywords: extract; analgesic activity; antioedema activity; nociception; acetic acid; formalin; carrageenan; dextran.

INTRODUCTION Justicia pectoralis Jacq., var. Stenophylla Leonard (Acanthaceae), is a medicinal herb largely utilized in the Northern and Northeastern regions of Brazil as an expectorant, antiasthmatic and analgesic. It is also used against cough and bronchitis and as an hallucinogen by the South American indians; in this case, probably because of the presence in its leaves of N,N,dimethyltryptamine (Melo and Andrade, 1989). De Vries et al., 1988, isolated several compounds from the extract of J. pectoralis, such as coumarin, 7-hydroxycoumarin (umbelliferone), dihydroxycoumarin and 3,2 hydroxyphenylpropionic acid. Other studies (Joseph et al., 1988a) isolated several types of avonoid compounds, and some of them were found for the rst time in species belonging to the Acanthaceae family. Five compounds were identied in the species grown at the city of Fortaleza, Brazil: 2H,1, benzopyranone- 2 (coumarin), 2H,7-hydroxy-1-benzopyranone-2 (umbelliferone), besides small amounts of ortho-hydroxy- transcinnamic acid (acetylated coumaric acid), ortho-hydroxydihydrocinnamic acetylated acid (acetylated melilotic acid) and also -sitosterol (Taveira, 1993). McCrae and Towers (1984), observed that the biological effects of J. pectoralis in respiratory diseases is associated with its antibacterial activity. In addition, the antiinammatory activity of the plant is probably due to the presence of coumarins (Mills et al., 1986) as well as the central and peripheral depressor effects (Mercier et al., 1953), the decrease in body temperature (Kitagawa, 1956; Kitagawa and Iwaki, 1958; Koh and Willoughby, 1979) and the vasodilator and antispasmodic activities (Sim oes et al., 1988). Other studies showed antitumour and antimitotic activities in coumarins extracted from the leaves of J. pectoralis (Murray et al.,

1982; Wall et al., 1988; Gawron and Glowniak, 1987; Rosskopf et al., 1992). The main objectives of the present paper were to study the analgesic and antiinammatory effects of the hydroalcohol extract from the leaves of J. pectoralis and its main constituents, coumarin and umbelliferone, in order to elucidate their mechanisms of action.

MATERIALS AND METHODS

Plant material. The aerial part of Justicia pectoralis, Jacq.,

var. Stenophylla Leon. (Acanthaceae), was collected at the Medicinal Plants Garden of Federal University of Cear a (Brazil). The exsicatae is deposited at the Prisco Bezerra Herbarium under the numbers 16,071 and 16,079.
Preparation of the hydroalcohol extract (HAE). One hundred

grams of fresh leaves from J. pectoralis was dried in the microwave oven at high intensity for 4.5 min, then extracted with 330 mL of ethyl alcohol and 660 mL of distilled water in the microwave oven for 15 min. The extract was ltered, placed in a dark ask and kept in the refrigerator. An aliquot (1 mL) was dried in the oven at 60C for the determination of solid residues. Before use, the alcohol was evaporated and the HAE concentrated as desired. The controls were treated with distilled water in the equivalent volume.
Preparation of the coumarin (CUM) and umbelliferone (UMB). The synthetic CUM, from Carlo Erba and the UMB

from Merck-Schuchardt Laboratories were dissolved in hot water and prepared, before use, at the desired concentration.
Writhing test. Male Swiss mice (2530 g) were used. The

Correspondence to: G. S. Viana. Contract grant sponsor: Brazilian National Research Council (CNPq). CCC 0951418X/97/03021105

animals were pretreated with the HAE (200 and 400 mg/kg,
Accepted 31 October 1996

1997 by John Wiley & Sons, Ltd.

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po), CUM (2.5 and 5.0 mg/kg, po), UMB (1.0 and 2.5 mg/ kg, po) or vehicle (distilled water: 0.1 mL/10 g, po). One hour later, they were injected with acetic acid 0.6% (0.1 mL/10 g) according to the method of Koster et al. (1959). Then, after 10 min, the number of writhings was registered for 20 min. The antinociceptive activity was evaluated in terms of the percentage of contraction inhibitions. In some experiments, naloxone (1 mg/kg, sc), an opioid antagonist, was administered 15 min before the treatment with the HAE, CUM, or UMB.
Formalin test. The formalin test was based on the method of

Table 1. Effects of HAE, CUM and UMB on the acetic acid induced in contraction mice
Group Dose No. of writhings Inhibition (%)

Control HAE HAE + NAL CUM CUM + NAL UMB UMB + NAL

Dubuisson and Dennis (1977) modied by Hunskaar and Hole (1985). Male Swiss mice (2530 g) were pretreated with HAE (200 and 400 mg/kg, po), CUM and UMB (2.5 and 5.0 mg/kg, po), morphine (MOR, 10 mg/kg, sc) or vehicle (1 mL distilled water/100 g, po). One hour or 30 min later, the animals were injected in the right hind paw with 20 L formalin 1% (0.1 mL/paw). Then, the animals were observed for the reaction to pain (licking time) during the rst phase (05 min), neurogenic, and the second phase (2025 min), inammatory. In some experiments, mice were pretreated with naloxone (1 mg/kg, sc) and, after 15 min, the animals received the HAE (400 mg/kg, po), CUM (5 mg/kg, po), UMB (5 mg/kg, po) and morphine (10 mg/kg, sc) or vehicle; 1 h or 30 min later, they were then injected with formalin. In other groups, mice were pretreated with L-arginine (150 and 300 mg/kg, ip) and, after 15 min, the animals received L-NAME 37 (5 mg/kg, ip) and UMB (5 mg/kg, po); 1 h or 30 min later, they were injected with formalin and observed during the rst and second phases of the formalin response.
Rat carrageenan oedema. Wistar rats of both sexes

200 mg/kg, po 400 mg/kg, po 400 mg/kg, po + 1.0 mg/kg,sc 2.5 mg/kg, po 5.0 mg/kg, po 5.0 mg/kg, po + 1.0 mg/kg, sc 1.0 mg/kg, po 2.5 mg/kg, po 2.5 mg/kg, po + 1.0 mg/kg, po

42.2 3.7 31.3 2.1a 17.6 1.0a 21.3 1.6a 30.0 2.2a 22.2 2.8a 19.0 4.0a 19.0 1.3a 11.0 3.9a 14.5 5.4a

25.0 42.0 49.5 29.0 46.0 55.0 74.0 65.6

Numbers represent mean SEM of 6 animals per group. Acetic acid 0.6% (0.1 mL/10 g) was injected intraperitoneally 1 h after the treatment with HAE, CUM and UMB. Naloxone was injected 15 min before the treatment with HAE, CUM and UMB. The number of contractions was registered 10 min after the acetic acid administration, during 20 min. a p < 0.05 (ANOVA and Fisher-test).

The results of the formalin test, also used to study antinociceptive effect, are presented in Table 2. The HAE (200 and 400 mg/kg, po), caused a similar inhibition (32%) of the rst phase, and 79% and 91% inhibition, respectively, of the second phase. CUM and UMB (2.5 and 5.0 mg/kg, po) caused inhibitions of the rst phase which ranged from 17% to 34%. Higher percentages of inhibition were seen in the second phase compared with the rst (CUM: 45% and 61.1%; UMB: 67% and 60.3% respectively). Again, the pretreatment with naloxone did not reverse the antinociception observed after the administration of HAE, CUM or
Table 2. Effects of HAE, CUM and UMB on the nociception induced by formalin in mice
Group Dose 1st phase 2nd phase

(150200 g) were pretreated, 1 h before the intraplantar injection of carrageenan 1% (0.1 mL/paw), with the HAE (200 and 400 mg/kg, po), CUM and UMB (20 mg/kg, po), indomethacin (INDO, 5 mg/kg, po) and vehicle (distilled water, 1 mL/100 g, po). The paw volume was measured with a plethysmometer (Ugo Basile, Italy), before and 1, 2, 3 and 4 h after the intraplantar injection of carrageenan (Winter et al., 1962).
Rat dextran oedema. Wistar rats of both sexes (150200 g)

Control HAE

200 mg/kg, po 400 mg/kg, po

HAE + NAL CUM

were pretreated with the HAE (50 mg/kg, po), CUM or UMB (20 mg/kg, po), cyproheptadine (CYPRO, 10 mg/kg, po) or vehicle (1 mL/100 g, po), 1 h before the injection of dextran 1.5% (0.1 mL in the right hind paw). The paw volume was measured with a plethysmometer (Ugo Basile, Italy), 30 min, 1, 2 and 3 h after the injection of dextran.

400 mg/kg, po + 1.0 mg/kg, sc 2.5 mg/kg, po 5.0 mg/kg, po

CUM + NAL UMB

5.0 mg/kg, po + 1.0 mg/kg, sc 2.5 mg/kg, po 5.0 mg/kg, po

RESULTS
UMB + NAL

Table 1 shows the effects of the HAE, CUM and UMB on the abdominal contractions produced by acetic acid in mice. It was demonstrated that HAE (200 and 400 mg/kg, po), inhibited the acetic acid contractions by 25% and 42%, while CUM (2.5 and 5.0 mg/kg, po) and UMB (1 and 2.5 mg/kg, po) caused inhibitions of the order of 29% and 45% and of 55% and 74%, respectively. Naloxone, an opioid antagonist, did not reverse the antinociceptive effect, suggesting the non-involvement of the opioid system in this effect, which in all cases was dose dependent and more intense in the group treated with UMB compared with CUM.

MOR MOR + NAL

5.0 mg/kg, po + 1.0 mg/kg, sc 10.0 mg/kg, sc 10.0 mg/kg, sc + 1.0 mg/kg, sc

54.5 2.5 (12) 37.0 2.0 (6)a (32.1%) 37.0 1.8 (6)a (32.1%) 36.0 5.2 (6)a (34.0%) 45.4 3.5(8) (16.7%) 42.0 5.8(6) (23.0%) 46.0 5.8(6) (15.6%) 44.6 5.2(8) (18.2%) 36.0 3.0(6)a (34.0%) 45.3 6.6(6) (16.4%) 19.3 4.2(6)a (64.6%) 49.0 4.2(6)b (10.1%)

25.2 2.2 5.3 3.9a (79%) 2.3 1.6a (91%) 3.5 1.3a (86.1%) 16.6 3.1a (34.1%) 9.8 3.1a (61.1%) 14.2 9.2 (43.6%) 8.4 2.8 (67.0%) 10.0 2.6 (60.3%) 17.6 8.7 (30.1%) 2.6 1.8a (89.7%) 9.3 5.4a (63.1%)

Numbers represent mean SEM of 6 animals per group. Formalin 1% (20 L/paw, sc) was injected in the mice hind paw, 1 h after treatment with the HAE, CUM, UMB or MOR. Naloxone was injected 15 min before treatment with the HAE, CUM, UMB or MOR. The licking time was registered from 05 min and from 2025 min after formalin. In parentheses, the percentage of inhibition. a p < 0.05 compared with controls, b p < 0.05 compared with MOR (ANOVA and Fisher-test).

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213

the rat paw volume at 2 h and 3 h in the carrageenan model compared with controls. The CUM and UMB at the same doses (10 and 20 mg/kg, po) were effective in decreasing the paw oedema at 3 h. The HAE and UMB did not produce an antioedematogenic effect with the dextran model. On the other hand, the CUM (20 mg/kg, po) was effective only at 2 h, causing a 22% inhibition.

DISCUSSION The results of the present paper showed that the HAE of J. pectoralis and its main constituents, coumarin and umbeliferone, possess analgesic and antiinammatory activities. The HAE, CUM and UMB signicantly inhibited pain produced by chemical stimuli such as acetic acid and formalin. The rst model is based on the application of a stimulus of high intensity, and the nociceptive response is of small duration. However, this test is nonspecic and the response is blocked by several classes of compounds, i.e. antihistaminic, hypotensor and CNS stimulant and depressor drugs (Hendershot and Forsaith, 1959). On the other hand, the formalin test is much more specic and largely used for studying nociception. This test permits evaluation of the animals response to a continuous pain of moderate intensity caused by the lesioned tissue, and the role of endogenous system in pain regulation (Tjolsen et al., 1992). The injection of formalin produces a biphasic response related to the pain process of early and late onset. The nociceptive stimulus increases the levels of substance P in the spinal cord and the initial increase, which occurs in the rst phase, is probably due to the direct activation of the population of peripheral nociceptors, while the decrease observed later could be due to the inactivation of substance P by formalin. The second phase corresponds to another increase in the levels of substance P due to a recruitment of inammatory cells by primary afferent neurons (McCarson and Goldstein, 1990). The antinociceptive effect of the HAE, CUM and UMB was not blocked by the opioid antagonist naloxone, suggesting the non-involvement of the opioid system. The mechanism of action is not clear but could be related to nitric oxide (NO). Nitric oxide is synthesized from Larginine by endothelial cells, occurring also in leucocytes,

Figure 1. Effect of L-arginine: 1 (150 mg/kg, po) and 2 (200 mg/ kg, po) on the analgesic activity of L-NAME 1 (37.5 mg/kg, po) and UMB (5 mg/kg, po) in the rst and second phases of nociception induced by formalin in mice. Numbers represent mean SEM of six rats per group. ap < 0.05 compared with controls; bp < 0.05 compared with L-NAME 1; cp < 0.05 compared with UMB (ANOVA and post hoc Fisher-test).

UMB. However, naloxone reversed as expected the antinociceptive effect demonstrated with morphine used as standard. In order to verify the possible involvement of the nitric oxide system in the antinociception produced by umbelliferone, the most active constituent of J. pectoralis, its effect was studied in the presence of L-arginine, known to block the nociception observed with L-NAME. L-arginine (150 and 300 mg/kg) antagonized the UMB effect on the second phase of the formalin test. The antagonism was complete in the group administered with the UMB and the higher dose of L-arginine, 300 mg/kg (Fig. 1). The antioedematogenic activities of HAE, CUM and UMB were also studied in the paw oedema models produced by carrageenan (Table 3) and dextran (Table 4) in rats. While no effect was seen with 200 mg/kg, po of the HAE, a higher dose (400 mg/kg, po) signicantly decreased

Table 3. Effect of HAE, CUM and UMB on the paw oedema induced by carrageenan in rats
Group Dose (mg/kg, po) 30 min Paw volume (mL) 1h 2h 3h

Control HAE

200 400 5 20 20

INDO Control CUM UMB

0.42 0.03 0.42 0.04 0.33 0.02 (26%) 0.31 0.07a (26%) 0.61 0.10 0.49 0.05 (20%) 0.43 0.04 (30%)

0.66 0.06 0.65 0.03 0.51 0.02a (23%) 0.38 0.04a (42%) 0.97 0.13 0.64 0.08 (34%) 0.68 0.11 (30%)

0.79 0.09 0.75 0.04 0.62 0.02a (22%) 0.44 0.06a (44%) 1.41 0.10 1.09 0.08a (23%) 0.88 0.07a (38%)

0.68 0.01 0.76 0.03 0.59 0.04 (13%) 0.43 0.06a (37%) 1.24 0.12 1.03 0.07 (17%) 0.79 0.09 (36%)

Numbers represent mean SEM of 6 rats per group. Animals were treated with the HAE, CUM, UMB and INDO, 1 h before the administration of carrageenan 1% (0.1 mL/paw). The paw volume was measured 1, 2, 3 and 4 h after carrageenan. a p < 0.05 compared with controls (ANOVA and Fishertest).

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Table 4. Effect of HAE, CUM, UMB and CYPRO on the paw oedema induced by dextran in rats
Group Dose (mg/kg, po) 30 min Paw volume (mL) 1h 2h 3h

Control HAE CUM UMB CYPRO

50 20 20 10

1.93 0.15 1.66 0.10 (14%) 1.86 0.09 (4%) 2.33 0.14 1.41 0.14a (27%)

2.35 0.13 1.97 0.15 (16%) 2.29 0.14 (3%) 2.67 0.11 1.55 0.12a (34%)

2.32 0.22 1.99 0.18 (14%) 1.81 0.13a (22%) 2.60 0.09 1.60 0.14a (31%)

1.98 0.11 1.71 0.15 (14%) 1.64 0.14 (17%) 2.43 0.01 1.62 0.16a (18%)

Numbers represent mean SEM of 6 rats per group. Animals were treated with the HAE, CUM, UMB and CYPRO, 1 h before the administration of dextran 1.5% (0.1 mL/paw). Paw volume was measured 1/2, 1, 2 and 3 h after the administration of dextran. a p < 0.05 compared with controls (ANOVA and Fisher-test).

Kupfer cells, hepatocytes, neuroblastoma, broblasts and the adrenal gland. It is responsible for the biological effects of the endothelial derived relaxing factor (EDRF). Nitric oxide stimulates the soluble guanylate cyclase in several tissues such as the brain, adrenal gland and platelets. It is thought that the compound can mediate vasodilation and other processes present in damaged tissues (Moore et al., 1991; Moncada, 1994). Agents which stimulate the synthesis of cGMP can produce a functional regulation concerning the decrease in nociceptors. Recent work showed that analgesics can produce a regulation in nociceptor sensitization through the stimulation of the L-arginine-nitric oxidecGMP path (Ferreira, 1994). The present work showed that L-arginine (substrate for the synthesis of nitric oxide) in mice (150 to 300 mg/kg, ip) completely reversed the antinociception in the second phase. L-NAME (which inhibits the nitric oxide synthesis) produced a signicant and predominant decrease of the nociception in the second phase, as seen by others, and it is possibly the result of a direct CNS action (Moore et al., 1991). Similarly, the effect of UMB (5 mg/kg, po) was also antagonized in a dose dependent way by L-arginine in the second phase, pointing to a role of nitric oxide in the observed analgesic effect of J. pectoralis. In previous work, (Rao et al., 1990), a potent antioedematogenic activity was also demonstrated with the HAE, CUM and UMB in the carrageenan model of acute inammation. However, in the dextran model, only the CUM were effective; HAE and UMB presented no effect. It

is known that coumarin acts on macrophages and stimulates phagocytosis (Koh and Willoughby, 1979), and its presence in the plant (MacCrae and Towers, 1984) explains the antiinammatory activity of J. pectoralis. As the HAE and UMB did not have any effect, it is possible that coumarin acts only on the inammatory response dependent on polymorphonuclear leucocytes. Carrageenan stimulates the release of several inammatory mediators such as histamine, serotonin, bradykinin and prostaglandins (Willoughby, 1975; Ohuchi et al., 1982). Non-steroidal antiinammatory drugs (NSAID) block synthesis of prostaglandins by inhibiting the enzyme cyclooxygenase. According to the results of the present work, the HAE, CUM and UMB seem to have a mechanism of action similar to NSAID, such as indomethacin and phenylbutazone, and the effect lasts for at least 3 h, time for the maximum effect of carrageenan (Crunkhorn and Meacock, 1972). On the other hand, only CUM was effective in the rat paw oedema induced by dextran, indicating that these substances can also act as membrane stabilizers especially on mastocytes, preventing their degradation and release of inammatory mediators such as histamine and serotonin. Surprisingly, the EHA and UMB did not show any effect in this model. Acknowledgements
We are grateful to Ms Vilani Rodrigues Bastos for technical assistance. The work had the nancial support of the Brazilian National Research Council (CNPq).

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