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Analgesic and antiinflammatory effects of chalcones isolated from Myracrodruon urundeuva Allemão
Phytomedicine 10: 189–195, 2003 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/phytomed
Phytomedicine
Analgesic and antiinflammatory effects of chalcones isolated from Myracrodruon urundeuva Allemão G. S. B. Viana, M. A. M. Bandeira, and F. J. A Matos Department of Physiology and Pharmacology, and Department of Organic Chemistry, Federal University of Ceará, Brazil
Summary The present work showed analgesic and antiinflammatory activities from a fraction containing three dimeric chalcones (chalcone enriched fraction – CEF), isolated from the stem-bark ethyl acetate extract of Myracrodruon urundeuva Allemão (Anacardiaceae). M. urundeuva is a popular medicinal plant used widely in Northeast Brazil, mainly as a topical female genital tract antiinflammatory. We observed that the CEF (5 and 10 mg/kg body wt., i.p. or p.o.) inhibited acetic acidinduced abdominal contractions in mice. In the formalin test, the CEF (5 and 10 mg/kg body wt.) was more effective intraperitoneally and inhibited predominantly the second phase of response. Naloxone reversed this effect, indicating an involvement of the opioid system. The CEF (10 and 20 mg/kg body wt.) also increased the reaction time to thermal stimuli in the hot-plate test in mice, after i.p. but not after p.o. administration. In the carrageenan-induced paw edema test in mice, the CEF (20 and 40 mg/kg body wt.) decreased paw volume significantly, after i.p. administration 2–4 hours after carrageenan injection. The CEF (40 mg/kg body wt.) was also active orally during the same period of time. The present work is the first report on peripheral and central analgesic effects and antiinflammatory activity of natural dimeric chalcones. Key words: Myracrodruon urundeuva, dimeric chalcones, analgesic and antiinflammatory effects
j Introduction Myracrodruon urundeuva Allemão (Anacardiaceae) is a 10- to 15-m high tree common in the Brazilian Northeastern “caatinga” (shrub forest), especially in Ceará state. It is also found in the Brazilian states of Bahia, Minas Gerais, and Goiás. An aqueous extract from the stem bark of this plant is used popularly in gynecology as a female genital tract antiinflammatory. The plant is also used in the treatment of respiratory and urinary tract diseases, hemoptysis, metrorrhagias and diarrheas, as an infusion or decoction, But is mostly used topically in post-partum gynecological treatment. It is also used in the treatment of skin wounds. Previous work (Menezes, 1986) showed that aqueous as well as hydroalcoholic extracts from M. urundeuva stem bark exert potent antiinflammatory activity
in experimental models of acute and sub-acute inflammation. The hydroalcoholic extract also showed hepatoprotective, anti-diarrheal and anti-ulcer activities (Morais et al., 1999). Chemical fractionation of the ethyl acetate extract yielded seven fractions, among which two showed pharmacological activity. One of them was a chalcone-enriched fraction (CEF), while the other presented catechic tannins as its main constituents. We have shown that both fractions are somewhat involved in the pharmacological activity of the plant (Viana et al., 1997). Chalcones and dihydrochalcones belong to the class of flavonoids which, apart from their antioxidant activity, are known for their ability to strengthen capillary walls, thus assisting circulation and helping to prevent 0944-7113/03/10/02-03-189 $ 15.00/0
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and treat bruising, varicose veins, bleeding gums and nosebleeds. They may also be useful in the treatment of heavy menstrual bleeding with no apparent cause. A third beneficial effect of some flavonoids, such as quercetin, rutin, curcumin, silymarin and green tea polyphenols, is their reputed antiinflammatory effect, which may be related to their ability to inhibit cyclooxygenase and lipooxygenase enzymes. Cyclooxygenase and lipooxygenase enzymes act on arachidonic acid metabolism in cell membranes to form potent inflammatory prostaglandins, some of which promote swelling and, possibly, symptoms such as headaches, rashes and joint pains (Zuanazzi, 1999). Since phytomedicines from M. urundeuva are popular remedies in Northeast Brazil, and considering that clinical studies (Melo et al., 1998) revealed an absence of significant toxic effects in the plant elixir, the objectives of the present work were to study analgesic and anti-inflammatory activities of chalcones isolated from the stem bark of M. urundeuva, in order to clarify their mechanism of action.
j Materials and Methods Plant Material
Myracrodruon urundeuva Allemão was originally collected near the city of Iguatu, Ceará, and identified by Dr. Afrânio Fernandes, of the Department of Biology of the Federal University of Ceará. It is presentely cultivated in the Medicinal Plant Garden of the same University. A voucher specimen is deposited at the Prisco Bezerra Herbarium under the number 14.999. Isolation of Chalcones
The chalcone-enriched fraction (CEF) was obtained from the ethyl acetate extract prepared with 5 kg of M. urundeuva ground stem bark, as described previonsly (Viana et al., 1995). The material was treated previously with hexane to remove lipid substances. The chromatographic fractionation of the ethyl acetate extract on a silica gel column resulted in seven fractions, after elution with chloroform, chroloform-acetone (9:1; 8:2; 7:3; 1:1), acetone, and methanol. Two fractions presented anti-inflammatory activity according to the pharmacological monitoring. Preliminary chemical tests showed that one fraction presented a predominance of chalcone type of compounds and the other mainly catechic tannins. The isolation of the chalcone type fraction compounds was performed using chromatographic techniques developed for that purpose (Bandeira, 2002), through the utilization of corn starch as the column support and elution with a mixture of dichloromethane/methanol (95:5). Through chromatographic procedure, three compounds were isolated
from that fraction (CEF). The analytical procedure used for structural identification of these compounds involved RMN 1H and RMN 13C (HBBD and DEPT θ = 135), with the contribution from bidimensional spectra of a homonuclear correlation of hydrogen and hydrogen (1H, 1H-COSY), as well as a heteronuclear correlation of hydrogen and carbon H, 13C-COSY-nJCH [n = 1 (HMQC) and n = 2 and n = 3, corresponding to HMBC and 1H, 1H-NOESY, respectively. The precise attribution of chemical displacements of carbon and hydrogen atoms revealed, for the first time, that the CEF is composed of three dimeric chalcones, named urundeuvine A, B and C, the chemical structures of which are shown in Figure 1. Pharmacological tests
Acetic acid-induced writhing: in this model (Koster et al., 1959), groups of 8–29 female mice each were administered 0.6% acetic acid (10 ml/kg body wt., i.p.), and the number of abdominal contractions was registered over 20 min, starting 10 min after acetic acid injection. Animals were treated intraperitoneally or orally with CEF (5 and 10 mg/kg body wt.), 30 or 60 min before acetic acid administration, respectively. Distilled water (vehicle) was used i.p. or p.o. in control animals. • Formalin test: in this test, licking time in seconds was registered from 0–5 min (first phase) and from 20–25 min (second phase), after the intraplantar administration of formalin (20 µl of a 1% v/v solution) in the right hind paw (Hunskaar et al., 1985; Hunskaar and Hole, 1987). A total of 102 female Swiss mice (20–25 g each) was treated intraperitoneally or orally with the CEF, 30 or 60 min before formalin injection, respectively. The possible involvement of the opioid receptor in CEF antinociceptive action was determined by the subcutaneous administration of naloxone (2 mg/kg body wt.), 15 min before the CEF (10 mg/kg body wt., i.p.) or morphine (5 mg/kg body wt., i.p.) injections, followed by the administration of formalin 30 min later. • Hot plate test: for the hot plate test (Eddy and Leimbach, 1953), groups of 9 to 18 female Swiss mice (20–25 g each) were treated with CEF (5–20 mg/kg body wt., i.p. or p.o.) or morphine (5 mg/kg body wt., i.p.) as standard. Measurements were performed before (0 time) and 30, 60 and 90 min after drug administration, with a cut-off time of 30 sec to avoid paw lesions. • Carrageenan-induced paw edema: a total of 80 female Swiss mice (20–25 g each) was treated with the intraplantar injection of carrageenan 1% (0.1 ml/paw) 30 or 60 min before the intraperitoneal or oral administration of CEF (5 to 40 mg/kg body wt., i.p. or p.o.), respectively. Indomethacin (10 mg/kg body wt., p.o.) and vehicle (distilled water, 1 ml/100 p.o. or i.p.) were also used as standard and control, respectively. Paw volume
Analgesic and antiinflammatory effects of chalcones was measured using a plethysmometer (Ugo Basile, Italy) before and 1, 2, 3 and 4 h after the intraplantar injection of carrageenan (Winter et al., 1962). • Statistical Analysis: all values are expressed as means ± SEM. Data were analyzed by ANOVA followed by Student-Newman-Keuls as the post hoc test. Results were considered statistically significant at p < 0.05.
j Results Table 1 shows the antinociceptive effect of the CEF on acetic acid-induced abdominal contractions in mice. The compounds caused 57 and 70% inhibition of contractions at doses of 5 and 10 mg/kg body wt., i.p., respectively. Oral administration was less efficient, and the absorption was somewhat irregular, producing 50 and 33% inhibitions at doses of 5 and 10 mg/kg body wt., respectively.
Fig. 1. Chemical structures of urundeuvines I, II and III chalcones isolated from M. urundeuva Allemão, as described in Methods.
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In the formalin test (Table 2), inhibition occurred predominantly during the second phase of the response. Thus, after i.p. administration of 5 and 10 mg/kg body wt. CEF caused a 76% response inhibition of the second phase. Pretreatment with naloxone partially but non-significantly reversed the CEF antinociceptive effect. As expected, morphine was very efficient during both the first (35% inhibition) and second (77% inhibition) phases and its effect was totally reversed by naloxone. Although 18 and 21% inhibition of the second phase was observed after the administration of 5 and 10 mg/kg body wt. of the CEF, this fraction was less efficient orally. In the hot-plate test (Table 3), although no significant effect was observed at CEF doses of 5 mg/kg body wt., i.p., 73 and 43% increases in the latency to thermal stimuli were observed at doses of 10 and 20 mg/kg body wt., i.p., after 30 and 60 min of drug administration, respectively. Increases of around 54 and 69%
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were also observed after administration at the dose of 20 mg/kg body wt., i.p., in the same observation periods. Interestingly, in both cases, the effect was long lasting, and 95 and 53% increases in thermal stimuli could still be observed 90 min after drug administration, at the doses of 10 and 20 mg/kg body wt., i.p., respectively. The CEF effect (10 mg/kg body wt., i.p.) was partially reversed by naloxone pretreatment after
Table 1. Effects of the chalcone enriched fraction (CEF) of Myracrodruon urundeuva Allemão on acetic acid-induced abdominal contractions in mice. Group
No. abdominal contractions/ 20 min
% inhibitions
Control, i.p. (29) CEF 5 mg/kg body wt., i.p. (13) 10 mg/kg body wt., i.p. (12)
42.6 ± 2.27
–
18.5 ± 2.40* 12.6 ± 2.66*
56.6 70.4
Control, p.o. (16) CEF 5 mg/kg body wt., p.o. (8) 10 mg/kg body wt., p.o. (12)
42.8 ± 2.33
–
21.4 ± 2.72* 28.8 ± 3.97*
50.0 33.0
Values are means ± SEM of the number of animals (in parentheses). Mice were treated with CEF 30 (i.p.) or 60 min (p.o.) before acetic acid injection. The number of contractions was measured for 20 min, 5 min after acetic acid administration. *p < 0.05 vs. control group.
Table 2. Effects of the chalcone enriched fraction (CEF) of Myracrodruon urundeuva Allemão in the formalin test in mice. Group
Licking Time (sec) –––st––––––––––––––––––––––––––––nd–––––––––––––––––––– 1 phase 2 phase
Control, i.p. (19) CEF 5 mg/kg body wt., i.p. (8) 10 mg/kg body wt., i.p. (12) Nal+CEF (7) Morphine, 5 mg/kg body wt., ip (7) Nal+Mor (7)
45.6 ± 17.8 18.8 ± 2.32
Control, p.o. (7) CEF 5 mg/kg body wt., p.o. (7) 10 mg/kg body wt., p.o. (7)
57.2 ± 8.13 16.4 ± 4.53
47.3 ± 2.49 48.7 ± 2.38 54.9 ± 5.92 29.7 ± 3.82* 60.1 ± 6.52
4.4 ± 1.73* 4.5 ± 1.82* 13.2 ± 4.19 4.4 ± 1.97* 13.6 ± 4.20
43.0 ± 4.04 20.6 ± 6.74 46.1 ± 4.45 14.9 ± 3.58
Values are means ± SEM of the number of animals (in parentheses). CEF was administered 30 min (i.p.) or 60 min (p.o.) before formalin injection, and licking time was measured as described in the Text. Nal – naloxone, 2 mg/kg body wt., s.c.; Mor – morphine, 5 mg/kg body wt., i.p. *p < 0.05 vs. control group.
30 min and reversed totally after 60 min of drug administration. No significant effect was observed after oral administration of CEF. The central analgesic effect of morphine was reversed totally by pretreatment with naloxone. The CEF also showed an antiinflammatory effect on the rat paw edema induced by carrageenan (Table 4), after oral and intraperitoneal administration, in a dosedependent manner. After intraperitoneal administration, significant results were observed at the highest dose, 40 mg/kg, at the third and fourth hour after carrageenan injection, with 48 and 35% reduction in paw volume, respectively. A small but significant decrease (22%) was also seen at the third hour, after administration of 10 mg/kg body wt., i.p. Significant antiinflammatory effects were also detected after the oral administration of 40 mg/kg body wt. of CEF, at the second, third and fourth hour, with decreases of paw volume on the order of 27, 59 and 50%, respectively. Indomethacin (10 mg/kg body wt., po) used as standard, decreased paw volume by 30% at the third hour.
j Discussion Flavonoids are a large group of naturally occurring compounds found in fruits, vegetables, grains, bark, roots, stems, flowers, tea and wine. A variety of in vitro and in vivo experiments have shown that selected flavonoids possess antiallergic, antiinflammatory, antiviral and antioxidant activities. Certain flavonoids possess potent inhibitory activity against a wide array of enzymes such as protein kinase C, protein tyrosine kinases, phospholipase A2 and others (Middleton, 1998). Other flavonoids (Manthey, 2000) potently inhibit prostaglandins, a group of powerful pro-inflammatory signaling molecules. Studies have shown that this effect is due to flavonoid inhibition of key enzymes involved in prostaglandin biosynthesis (i.e., lipoxygenase, phospholipase, and cyclooxygenase). Flavonoids also inhibit phosphodiesterases involved in cell activation. Much of this effect is upon the biosynthesis of protein cytokines that mediate adhesion of circulating leukocytes to sites of injury. Protein kinases are another class of regulatory enzymes affected by flavonoids. Inhibition of these key enzymes provides the mechanism by which flavonoids inhibit inflammatory processes (Manthey et al., 2001). In addition, the deleterious effects of excessive release of nitric oxide (NO) have been implicated in tissue damage and inflammation. Recent results (Srivastava et al., 2000) suggest that tannic acid and polyphenols are potent inhibitors of NO synthase activity and NO production, and that these effects are independent of their antioxidant activity.
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Table 3. Effects of the chalcone enriched fraction (CEF) of Myracrodruon urundeuva Allemão in the hot-plate test in mice. Group
T0
T30
T60
T90
Control (8) CEF 5 mg/kg body wt., i.p. (9) 10 mg/kg body wt., i.p. (9) 20 mg/kg body wt.,.i.p. (10) Nal+CEF,10 mg/kg body wt., ip Mor 5 mg/kg body wt., i.p. (9) Nal+Mor (9) CEF 10 mg/kg body wt., p.o. (9) 20 mg/kg body wt.,p.o. (9)
14.3 ± 1.35
13.0 ± 1.01
12.7 ± 0.76
11.7 ± 0.94
13.1 ± 1.56 15.6 ± 1.29 13.1 ± 1.05 13.6 ± 1.36 16.3 ± 2.27 13.8 ± 2.14
15.5 ± 2.09 22.5 ± 3.76* 20.0 ± 2.04* 17.9 ± 2.50 35.4 ± 2.30* 12.1 ± 1.64
12.5 ± 1.33 18.1 ± 3.16* 21.5 ± 1.78* 14.8 ± 1.82 30.7 ± 3.38* 11.5 ± 1.27
12.5 ± 1.16 22.4 ± 1.61* 17.9 ± 2.28* 8.1 ± 1.60 20.7 ± 2.80* 13.3 ± 2.15
13.4 ± 1.85 10.3 ± 1.14
15.1 ± 1.23 11.9 ± 1.27
11.4 ± 1.88 12.9 ± 0.97
12.5 ± 1.27 10.4 ± 1.04
Values are means ± SEM of the number of animals (in parentheses). Latencies to thermal stimuli were measured at several times (immediately (T0), and at 30 (T30), 60 (T60) and 90 (T90) min), 30 min (i.p.) or 60 min (p.o.) after drug administration. Mor – morphine; Nal – naloxone, 2 mg/kg body wt., s.c. *p < 0.05 vs. control group at the same period of time.
Table 4. Effect of the chalcone enriched fraction (CEF) of Myracrodruon urundeuva Allemão in carrageenan-induced paw edema in mice. Group
Paw volume (ml) at the ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 1st hour 2nd hour 3rd hour 4th hour
Control, i.p. (22) CEF 5 mg/kg body wt., i.p. (8) 10 mg/kg body wt., i.p. (14) 40 mg/kg body wt., i.p. (8) CEF 10 mg/kg body wt., p.o. (6) 20 mg/kg body wt., p.o. (6) 40 mg/kg body wt., p.o. (8) Indomethacin 10 mg/kg body wt., p.o. (6)
0.15 ± 0.01
0.20 ± 0.013
0.27 ± 0.013
0.22 ± 0.014
0.15 ± 0.015 0.14 ± 0.013 0.11 ± 0.014
0.20 ± 0.009 0.19 ± 0.011 0.18 ± 0.016
0.22 ± 0.01 0.21 ± 0.013* 0.14 ± 0.013*
0.17 ± 0.016 0.17 ± 0.013 0.13 ± 0.014*
0.19 ± 0.016 0.16 ± 0.013 0.12 ± 0.012
0.26 ± 0.024 0.23 ± 0.023 0.16 ± 0.009
0.33 ± 0.024 0.23 ± 0.013 0.11 ± 0.009*
0.28 ± 0.018 0.21 ± 0.021 0.10 ± 0.009*
0.10 ± 0.011
0.20 ± 0.013
0.19 ± 0.02*
0.18 ± 0.018*
Values are means ± SEM of the number of animals in parentheses. CEF was administered 30 (i.p.) or 60 min (p.o.) before carrageenan injection. Edema volume was measured before and at 1, 2, 3 and 4 h after carrageenan injection. Control – carrageenan alone. *p < 0.05 vs. control group over the same period of time.
Chalcones isolated from natural products are known to possess several important biological properties, including antifungal (ElSohly et al., 2001; Lopez et al., 2001), leishmanicidal (Kayser and Kiderlen, 2001; Chen et al., 2001; Nielsen et al, 1998), and antimalarial (Dominguez er al., 2001; Ram et al., 2000; Li et al., 1995). Some other researchers also discuss the ability of chalcones to inhibit lipid peroxidation in rat liver microsomes (Rodriguez et al., 2001; Cos et al., 2001) and mouse liver (Machala et al., 2001) and to inhibit in vitro oxidation of low-density lipoproteins (Miranda et al., 2000). Another recent study (Phan et al., 2001) showed that several compounds including chalcones
isolated from Chromolaena odorata are powerful antioxidants, protecting cultured skin cells against oxidative damage. It has been shown that reactive oxygen and nitrogen species contribute to the pathophysiology of inflammatory conditions. A recent report indicates that a chalcone derivative inhibited chemiluminescence in vitro, cell migration and eicosanoid and TNF-alpha in mouse air pouch injected with zymosan. This chalcone derivative also exerted inflammatory effects in the carrageenan paw edema (Herencia et al., 2001). Hsieh et al. (1998) showed that 2′,5′-dihydroxychalcone may be a promising antiinflammatory agent,
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because it is a potent mediator and cyclooxygenase inhibitor. Their results indicated that the antiinflammatory effects of these compounds are mediated, at least partly, through the suppression of chemical mediators released from mast cells and neutrophils. Recently, Nakamura et al. (2002) synthesized fluorinated 3,4-dihydroxychalcones which were shown to be 5-lipoxygenase inhibitors and inhibitors of peroxidation in rat liver microsomes. Correa et al. (2001) synthesized eleven chalcones which were tested for their antinociceptive activity using the writhing test in mice. The authors showed that some of these compounds, after intraperitoneal administration, caused potent and dose-related antinociception. Among the compounds tested, 3,4-dichlorochalcone was the most effective one and, in the formalin test, inhibited only the inflammatory pain (second phase of the test). In the present work, we studied antinociceptive and antiedematogenic activities of a chalcone-enriched fraction (CEF) isolated from M. urundeuva, a medicinal plant largely used in Northeast Brazil for the treatment of several diseases, especially as an antiinflammatory in gynecological disorders. We showed a significant and dose-dependent antinociceptive effect of chalcones at low doses, after intraperitoneal administration, in the writhing test in mice. These compounds were less effective orally, probably due to a somewhat erratic absorption. In the formalin test, which measures pain of both neurogenic (first phase) and inflammatory (second phase) origins, the CEF inhibited preferentially the second phase of the response, and the opioid system seems to have a role in this effect. A small but significant decrease in carrageenan-induced paw edema was also detected, after intraperitoneal as well as after oral administration of a higher dose. Antiinflammatory effects have already been demonstrated with chalcones (Hsieh et al., 1998), and some chalcone derivatives have been reported to be potent chemical mediators and cyclooxygenase inhibitors. Hsieh et al. (1998) showed that some chalcones present strong inhibitory effects on the release of β-glucuronidase and histamine from rat peritoneal mast cells stimulated with compound 48/80. Other chalcones exhibited potent inhibitory effects on the release of β-glucuronidase and lysozyme from rat neutrophils stimulated with fMLP. Also, Lin et al. (1997) tested some chalcone derivatives for their inhibitory effects on platelet aggregation and activation of mast cells and neutrophils. They reported that the arachidonic acid-induced platelet aggregation was inhibited potently by almost all those compounds, and some also showed a potent inhibitory effect on collagen-induced platelet aggregation and cyclooxygenase. The authors suggest
that the anti-platelet effects of chalcones are mainly a result of the inhibition of thromboxane formation. It is possible that at least part of the antiinflammatory effect observed in the present work may also be due to platelet anti-aggregation, as we observed that CEF inhibited around 20% of ADP-induced-aggregation in human platelets (data not shown). Analgesic and anti-inflammatory effects have already been observed in flavonoids as well as in tannins (Ahmadiani et al., 1998; Ahmadiani et al., 2000). In the present work, the observed CEF antinociceptive effect is probably related to the opioid system. Although antiinflammatory effects have also been detected in chalcones, the present results are, as far as we know, the first report of peripheral and central analgesic as well as antiinflammatory effects of dimeric chalcones from Myracrodruon urundeuva. Acknowledgments
Authors are grateful to the technical assistance of Ms. M. Vilani Rodrigues Bastos. The work had the financial support of the Brazilian National Research Council (CNPq) and the Research Support Foundation (FUNCAP) of the State of Ceará, Brazil.
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Miranda CL, Stevens JF, Ivanov V, McCall M, Frei B, Deinzer ML, Buhler DR (2000) Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. J Agric Food Chem 48: 3876–3884 Morais MM, Belarmino-Filho JN, Nery EA, Viana GSB, Brito GAC, Ribeiro RA (1999) Comparação dos efeitos de xtrato hidroalcoólico de Myracrodruon urundeuva Allemão e do MESNA na patogênese da cistite hemorrágica induzida por ifosfamida. XIV Reunião Annual da FESBE, Caxambu, MG, Brasil Nakamura C, Kawasaki N, Miyataka H, Jayachandran E, Kim IH, Kirk KL, Taguchi T, Takeuchi Y, Hori H, Satoh T (2002) Synthesis and biological activities of fluorinated chalcone derivatives. Bioorg Med Chem 10: 699–706 Nielsen SF, Christensen SB, Cruciani G, Kharazani A, Liljefors T (1998) Antileishmanial chalcones: statistical design, synthesis, and three-dimensional quantitative structure-activity relationshipn analysis. J Med Chem 41: 4819–4832 Phan TT, Wang L, See P, Grayer RJ, Chan SY, Lee ST (2001) Phenolic compounds of Chromolaena odorata protect cultured skin cells from oxidative damage: implication for cutaneous wound healing. Biol Pharm Bull 24: 1373–1379 Ram VJ, Saxena AS, Srivastava S, Chandra S (2001) Oxygenated chalcones and bischalcones as potential antimalarial agents. Bioorg Med Chem 10: 2159–2161 Rodriguez RJ, Miranda CL, Stevens JF, Deinzer ML, Buhler DR (2001) Influence of prenylated and non-prenylated flavonoids on liver microsomal lipid peroxidation and oxidative injury in rat hepatocytes. Food Chem Toxicol 39: 437–445 Srivastava RC, Hussain MM, Hasan SK, Athar M (2000) Green tea polyphenols and tannic acid act as potent inhibitors of phorbol ester-induced nitric oxide generation in rat hepatocytes independent of their antioxidant properties. Cancer Lett 153: 1–5 Viana GSB, Bandeira MAM, Moura LC, Souza-Filho MVP, Matos FJA, Ribeiro RA (1997) Analgesic and antiinflammatory effects of the tannin fraction from Myracrodruon urundeuva Allemão. Phytother Res 11: 118–122 Viana GSB, Matos FJA, Bandeira MAM, Rao VSN (1995) Aroeira-do-Sertão (Myracrodruon urundeuva Allemão): Estudo Botânico, Farmacognóstico, Químico e Farmacológico, Fortaleza, Brasil, EUFC Winter CA, Riseley EA, Nuss GW (1962) Carrageenan-induced edema in the hind paw of the rats as an assay for antiinflammatory drugs. Proc Soc Exp Biol Med 111: 544–547 Zuanazzi JAS (1999) Flavonóides. In: Farmacognosia, da planta ao medicamento (Cláudia MO Simões, Eloir P Schenkel, Grace Grosmann, João Carlos P Melo, Lilian A Mentz, Pedro R Petrovick (eds). UFRGS, p. 489–516
j Address G. Viana, Federal University of Ceará, Rua Barbosa de Freitas, 130/1100, 60.170-020 Fortaleza, Brazil Fax: ++55-85-242-3064; e-mail: [email protected]
Phytomedicine
Analgesic and antiinflammatory effects of chalcones isolated from Myracrodruon urundeuva Allemão G. S. B. Viana, M. A. M. Bandeira, and F. J. A Matos Department of Physiology and Pharmacology, and Department of Organic Chemistry, Federal University of Ceará, Brazil
Summary The present work showed analgesic and antiinflammatory activities from a fraction containing three dimeric chalcones (chalcone enriched fraction – CEF), isolated from the stem-bark ethyl acetate extract of Myracrodruon urundeuva Allemão (Anacardiaceae). M. urundeuva is a popular medicinal plant used widely in Northeast Brazil, mainly as a topical female genital tract antiinflammatory. We observed that the CEF (5 and 10 mg/kg body wt., i.p. or p.o.) inhibited acetic acidinduced abdominal contractions in mice. In the formalin test, the CEF (5 and 10 mg/kg body wt.) was more effective intraperitoneally and inhibited predominantly the second phase of response. Naloxone reversed this effect, indicating an involvement of the opioid system. The CEF (10 and 20 mg/kg body wt.) also increased the reaction time to thermal stimuli in the hot-plate test in mice, after i.p. but not after p.o. administration. In the carrageenan-induced paw edema test in mice, the CEF (20 and 40 mg/kg body wt.) decreased paw volume significantly, after i.p. administration 2–4 hours after carrageenan injection. The CEF (40 mg/kg body wt.) was also active orally during the same period of time. The present work is the first report on peripheral and central analgesic effects and antiinflammatory activity of natural dimeric chalcones. Key words: Myracrodruon urundeuva, dimeric chalcones, analgesic and antiinflammatory effects
j Introduction Myracrodruon urundeuva Allemão (Anacardiaceae) is a 10- to 15-m high tree common in the Brazilian Northeastern “caatinga” (shrub forest), especially in Ceará state. It is also found in the Brazilian states of Bahia, Minas Gerais, and Goiás. An aqueous extract from the stem bark of this plant is used popularly in gynecology as a female genital tract antiinflammatory. The plant is also used in the treatment of respiratory and urinary tract diseases, hemoptysis, metrorrhagias and diarrheas, as an infusion or decoction, But is mostly used topically in post-partum gynecological treatment. It is also used in the treatment of skin wounds. Previous work (Menezes, 1986) showed that aqueous as well as hydroalcoholic extracts from M. urundeuva stem bark exert potent antiinflammatory activity
in experimental models of acute and sub-acute inflammation. The hydroalcoholic extract also showed hepatoprotective, anti-diarrheal and anti-ulcer activities (Morais et al., 1999). Chemical fractionation of the ethyl acetate extract yielded seven fractions, among which two showed pharmacological activity. One of them was a chalcone-enriched fraction (CEF), while the other presented catechic tannins as its main constituents. We have shown that both fractions are somewhat involved in the pharmacological activity of the plant (Viana et al., 1997). Chalcones and dihydrochalcones belong to the class of flavonoids which, apart from their antioxidant activity, are known for their ability to strengthen capillary walls, thus assisting circulation and helping to prevent 0944-7113/03/10/02-03-189 $ 15.00/0
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and treat bruising, varicose veins, bleeding gums and nosebleeds. They may also be useful in the treatment of heavy menstrual bleeding with no apparent cause. A third beneficial effect of some flavonoids, such as quercetin, rutin, curcumin, silymarin and green tea polyphenols, is their reputed antiinflammatory effect, which may be related to their ability to inhibit cyclooxygenase and lipooxygenase enzymes. Cyclooxygenase and lipooxygenase enzymes act on arachidonic acid metabolism in cell membranes to form potent inflammatory prostaglandins, some of which promote swelling and, possibly, symptoms such as headaches, rashes and joint pains (Zuanazzi, 1999). Since phytomedicines from M. urundeuva are popular remedies in Northeast Brazil, and considering that clinical studies (Melo et al., 1998) revealed an absence of significant toxic effects in the plant elixir, the objectives of the present work were to study analgesic and anti-inflammatory activities of chalcones isolated from the stem bark of M. urundeuva, in order to clarify their mechanism of action.
j Materials and Methods Plant Material
Myracrodruon urundeuva Allemão was originally collected near the city of Iguatu, Ceará, and identified by Dr. Afrânio Fernandes, of the Department of Biology of the Federal University of Ceará. It is presentely cultivated in the Medicinal Plant Garden of the same University. A voucher specimen is deposited at the Prisco Bezerra Herbarium under the number 14.999. Isolation of Chalcones
The chalcone-enriched fraction (CEF) was obtained from the ethyl acetate extract prepared with 5 kg of M. urundeuva ground stem bark, as described previonsly (Viana et al., 1995). The material was treated previously with hexane to remove lipid substances. The chromatographic fractionation of the ethyl acetate extract on a silica gel column resulted in seven fractions, after elution with chloroform, chroloform-acetone (9:1; 8:2; 7:3; 1:1), acetone, and methanol. Two fractions presented anti-inflammatory activity according to the pharmacological monitoring. Preliminary chemical tests showed that one fraction presented a predominance of chalcone type of compounds and the other mainly catechic tannins. The isolation of the chalcone type fraction compounds was performed using chromatographic techniques developed for that purpose (Bandeira, 2002), through the utilization of corn starch as the column support and elution with a mixture of dichloromethane/methanol (95:5). Through chromatographic procedure, three compounds were isolated
from that fraction (CEF). The analytical procedure used for structural identification of these compounds involved RMN 1H and RMN 13C (HBBD and DEPT θ = 135), with the contribution from bidimensional spectra of a homonuclear correlation of hydrogen and hydrogen (1H, 1H-COSY), as well as a heteronuclear correlation of hydrogen and carbon H, 13C-COSY-nJCH [n = 1 (HMQC) and n = 2 and n = 3, corresponding to HMBC and 1H, 1H-NOESY, respectively. The precise attribution of chemical displacements of carbon and hydrogen atoms revealed, for the first time, that the CEF is composed of three dimeric chalcones, named urundeuvine A, B and C, the chemical structures of which are shown in Figure 1. Pharmacological tests
Acetic acid-induced writhing: in this model (Koster et al., 1959), groups of 8–29 female mice each were administered 0.6% acetic acid (10 ml/kg body wt., i.p.), and the number of abdominal contractions was registered over 20 min, starting 10 min after acetic acid injection. Animals were treated intraperitoneally or orally with CEF (5 and 10 mg/kg body wt.), 30 or 60 min before acetic acid administration, respectively. Distilled water (vehicle) was used i.p. or p.o. in control animals. • Formalin test: in this test, licking time in seconds was registered from 0–5 min (first phase) and from 20–25 min (second phase), after the intraplantar administration of formalin (20 µl of a 1% v/v solution) in the right hind paw (Hunskaar et al., 1985; Hunskaar and Hole, 1987). A total of 102 female Swiss mice (20–25 g each) was treated intraperitoneally or orally with the CEF, 30 or 60 min before formalin injection, respectively. The possible involvement of the opioid receptor in CEF antinociceptive action was determined by the subcutaneous administration of naloxone (2 mg/kg body wt.), 15 min before the CEF (10 mg/kg body wt., i.p.) or morphine (5 mg/kg body wt., i.p.) injections, followed by the administration of formalin 30 min later. • Hot plate test: for the hot plate test (Eddy and Leimbach, 1953), groups of 9 to 18 female Swiss mice (20–25 g each) were treated with CEF (5–20 mg/kg body wt., i.p. or p.o.) or morphine (5 mg/kg body wt., i.p.) as standard. Measurements were performed before (0 time) and 30, 60 and 90 min after drug administration, with a cut-off time of 30 sec to avoid paw lesions. • Carrageenan-induced paw edema: a total of 80 female Swiss mice (20–25 g each) was treated with the intraplantar injection of carrageenan 1% (0.1 ml/paw) 30 or 60 min before the intraperitoneal or oral administration of CEF (5 to 40 mg/kg body wt., i.p. or p.o.), respectively. Indomethacin (10 mg/kg body wt., p.o.) and vehicle (distilled water, 1 ml/100 p.o. or i.p.) were also used as standard and control, respectively. Paw volume
Analgesic and antiinflammatory effects of chalcones was measured using a plethysmometer (Ugo Basile, Italy) before and 1, 2, 3 and 4 h after the intraplantar injection of carrageenan (Winter et al., 1962). • Statistical Analysis: all values are expressed as means ± SEM. Data were analyzed by ANOVA followed by Student-Newman-Keuls as the post hoc test. Results were considered statistically significant at p < 0.05.
j Results Table 1 shows the antinociceptive effect of the CEF on acetic acid-induced abdominal contractions in mice. The compounds caused 57 and 70% inhibition of contractions at doses of 5 and 10 mg/kg body wt., i.p., respectively. Oral administration was less efficient, and the absorption was somewhat irregular, producing 50 and 33% inhibitions at doses of 5 and 10 mg/kg body wt., respectively.
Fig. 1. Chemical structures of urundeuvines I, II and III chalcones isolated from M. urundeuva Allemão, as described in Methods.
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In the formalin test (Table 2), inhibition occurred predominantly during the second phase of the response. Thus, after i.p. administration of 5 and 10 mg/kg body wt. CEF caused a 76% response inhibition of the second phase. Pretreatment with naloxone partially but non-significantly reversed the CEF antinociceptive effect. As expected, morphine was very efficient during both the first (35% inhibition) and second (77% inhibition) phases and its effect was totally reversed by naloxone. Although 18 and 21% inhibition of the second phase was observed after the administration of 5 and 10 mg/kg body wt. of the CEF, this fraction was less efficient orally. In the hot-plate test (Table 3), although no significant effect was observed at CEF doses of 5 mg/kg body wt., i.p., 73 and 43% increases in the latency to thermal stimuli were observed at doses of 10 and 20 mg/kg body wt., i.p., after 30 and 60 min of drug administration, respectively. Increases of around 54 and 69%
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were also observed after administration at the dose of 20 mg/kg body wt., i.p., in the same observation periods. Interestingly, in both cases, the effect was long lasting, and 95 and 53% increases in thermal stimuli could still be observed 90 min after drug administration, at the doses of 10 and 20 mg/kg body wt., i.p., respectively. The CEF effect (10 mg/kg body wt., i.p.) was partially reversed by naloxone pretreatment after
Table 1. Effects of the chalcone enriched fraction (CEF) of Myracrodruon urundeuva Allemão on acetic acid-induced abdominal contractions in mice. Group
No. abdominal contractions/ 20 min
% inhibitions
Control, i.p. (29) CEF 5 mg/kg body wt., i.p. (13) 10 mg/kg body wt., i.p. (12)
42.6 ± 2.27
–
18.5 ± 2.40* 12.6 ± 2.66*
56.6 70.4
Control, p.o. (16) CEF 5 mg/kg body wt., p.o. (8) 10 mg/kg body wt., p.o. (12)
42.8 ± 2.33
–
21.4 ± 2.72* 28.8 ± 3.97*
50.0 33.0
Values are means ± SEM of the number of animals (in parentheses). Mice were treated with CEF 30 (i.p.) or 60 min (p.o.) before acetic acid injection. The number of contractions was measured for 20 min, 5 min after acetic acid administration. *p < 0.05 vs. control group.
Table 2. Effects of the chalcone enriched fraction (CEF) of Myracrodruon urundeuva Allemão in the formalin test in mice. Group
Licking Time (sec) –––st––––––––––––––––––––––––––––nd–––––––––––––––––––– 1 phase 2 phase
Control, i.p. (19) CEF 5 mg/kg body wt., i.p. (8) 10 mg/kg body wt., i.p. (12) Nal+CEF (7) Morphine, 5 mg/kg body wt., ip (7) Nal+Mor (7)
45.6 ± 17.8 18.8 ± 2.32
Control, p.o. (7) CEF 5 mg/kg body wt., p.o. (7) 10 mg/kg body wt., p.o. (7)
57.2 ± 8.13 16.4 ± 4.53
47.3 ± 2.49 48.7 ± 2.38 54.9 ± 5.92 29.7 ± 3.82* 60.1 ± 6.52
4.4 ± 1.73* 4.5 ± 1.82* 13.2 ± 4.19 4.4 ± 1.97* 13.6 ± 4.20
43.0 ± 4.04 20.6 ± 6.74 46.1 ± 4.45 14.9 ± 3.58
Values are means ± SEM of the number of animals (in parentheses). CEF was administered 30 min (i.p.) or 60 min (p.o.) before formalin injection, and licking time was measured as described in the Text. Nal – naloxone, 2 mg/kg body wt., s.c.; Mor – morphine, 5 mg/kg body wt., i.p. *p < 0.05 vs. control group.
30 min and reversed totally after 60 min of drug administration. No significant effect was observed after oral administration of CEF. The central analgesic effect of morphine was reversed totally by pretreatment with naloxone. The CEF also showed an antiinflammatory effect on the rat paw edema induced by carrageenan (Table 4), after oral and intraperitoneal administration, in a dosedependent manner. After intraperitoneal administration, significant results were observed at the highest dose, 40 mg/kg, at the third and fourth hour after carrageenan injection, with 48 and 35% reduction in paw volume, respectively. A small but significant decrease (22%) was also seen at the third hour, after administration of 10 mg/kg body wt., i.p. Significant antiinflammatory effects were also detected after the oral administration of 40 mg/kg body wt. of CEF, at the second, third and fourth hour, with decreases of paw volume on the order of 27, 59 and 50%, respectively. Indomethacin (10 mg/kg body wt., po) used as standard, decreased paw volume by 30% at the third hour.
j Discussion Flavonoids are a large group of naturally occurring compounds found in fruits, vegetables, grains, bark, roots, stems, flowers, tea and wine. A variety of in vitro and in vivo experiments have shown that selected flavonoids possess antiallergic, antiinflammatory, antiviral and antioxidant activities. Certain flavonoids possess potent inhibitory activity against a wide array of enzymes such as protein kinase C, protein tyrosine kinases, phospholipase A2 and others (Middleton, 1998). Other flavonoids (Manthey, 2000) potently inhibit prostaglandins, a group of powerful pro-inflammatory signaling molecules. Studies have shown that this effect is due to flavonoid inhibition of key enzymes involved in prostaglandin biosynthesis (i.e., lipoxygenase, phospholipase, and cyclooxygenase). Flavonoids also inhibit phosphodiesterases involved in cell activation. Much of this effect is upon the biosynthesis of protein cytokines that mediate adhesion of circulating leukocytes to sites of injury. Protein kinases are another class of regulatory enzymes affected by flavonoids. Inhibition of these key enzymes provides the mechanism by which flavonoids inhibit inflammatory processes (Manthey et al., 2001). In addition, the deleterious effects of excessive release of nitric oxide (NO) have been implicated in tissue damage and inflammation. Recent results (Srivastava et al., 2000) suggest that tannic acid and polyphenols are potent inhibitors of NO synthase activity and NO production, and that these effects are independent of their antioxidant activity.
Analgesic and antiinflammatory effects of chalcones
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Table 3. Effects of the chalcone enriched fraction (CEF) of Myracrodruon urundeuva Allemão in the hot-plate test in mice. Group
T0
T30
T60
T90
Control (8) CEF 5 mg/kg body wt., i.p. (9) 10 mg/kg body wt., i.p. (9) 20 mg/kg body wt.,.i.p. (10) Nal+CEF,10 mg/kg body wt., ip Mor 5 mg/kg body wt., i.p. (9) Nal+Mor (9) CEF 10 mg/kg body wt., p.o. (9) 20 mg/kg body wt.,p.o. (9)
14.3 ± 1.35
13.0 ± 1.01
12.7 ± 0.76
11.7 ± 0.94
13.1 ± 1.56 15.6 ± 1.29 13.1 ± 1.05 13.6 ± 1.36 16.3 ± 2.27 13.8 ± 2.14
15.5 ± 2.09 22.5 ± 3.76* 20.0 ± 2.04* 17.9 ± 2.50 35.4 ± 2.30* 12.1 ± 1.64
12.5 ± 1.33 18.1 ± 3.16* 21.5 ± 1.78* 14.8 ± 1.82 30.7 ± 3.38* 11.5 ± 1.27
12.5 ± 1.16 22.4 ± 1.61* 17.9 ± 2.28* 8.1 ± 1.60 20.7 ± 2.80* 13.3 ± 2.15
13.4 ± 1.85 10.3 ± 1.14
15.1 ± 1.23 11.9 ± 1.27
11.4 ± 1.88 12.9 ± 0.97
12.5 ± 1.27 10.4 ± 1.04
Values are means ± SEM of the number of animals (in parentheses). Latencies to thermal stimuli were measured at several times (immediately (T0), and at 30 (T30), 60 (T60) and 90 (T90) min), 30 min (i.p.) or 60 min (p.o.) after drug administration. Mor – morphine; Nal – naloxone, 2 mg/kg body wt., s.c. *p < 0.05 vs. control group at the same period of time.
Table 4. Effect of the chalcone enriched fraction (CEF) of Myracrodruon urundeuva Allemão in carrageenan-induced paw edema in mice. Group
Paw volume (ml) at the ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 1st hour 2nd hour 3rd hour 4th hour
Control, i.p. (22) CEF 5 mg/kg body wt., i.p. (8) 10 mg/kg body wt., i.p. (14) 40 mg/kg body wt., i.p. (8) CEF 10 mg/kg body wt., p.o. (6) 20 mg/kg body wt., p.o. (6) 40 mg/kg body wt., p.o. (8) Indomethacin 10 mg/kg body wt., p.o. (6)
0.15 ± 0.01
0.20 ± 0.013
0.27 ± 0.013
0.22 ± 0.014
0.15 ± 0.015 0.14 ± 0.013 0.11 ± 0.014
0.20 ± 0.009 0.19 ± 0.011 0.18 ± 0.016
0.22 ± 0.01 0.21 ± 0.013* 0.14 ± 0.013*
0.17 ± 0.016 0.17 ± 0.013 0.13 ± 0.014*
0.19 ± 0.016 0.16 ± 0.013 0.12 ± 0.012
0.26 ± 0.024 0.23 ± 0.023 0.16 ± 0.009
0.33 ± 0.024 0.23 ± 0.013 0.11 ± 0.009*
0.28 ± 0.018 0.21 ± 0.021 0.10 ± 0.009*
0.10 ± 0.011
0.20 ± 0.013
0.19 ± 0.02*
0.18 ± 0.018*
Values are means ± SEM of the number of animals in parentheses. CEF was administered 30 (i.p.) or 60 min (p.o.) before carrageenan injection. Edema volume was measured before and at 1, 2, 3 and 4 h after carrageenan injection. Control – carrageenan alone. *p < 0.05 vs. control group over the same period of time.
Chalcones isolated from natural products are known to possess several important biological properties, including antifungal (ElSohly et al., 2001; Lopez et al., 2001), leishmanicidal (Kayser and Kiderlen, 2001; Chen et al., 2001; Nielsen et al, 1998), and antimalarial (Dominguez er al., 2001; Ram et al., 2000; Li et al., 1995). Some other researchers also discuss the ability of chalcones to inhibit lipid peroxidation in rat liver microsomes (Rodriguez et al., 2001; Cos et al., 2001) and mouse liver (Machala et al., 2001) and to inhibit in vitro oxidation of low-density lipoproteins (Miranda et al., 2000). Another recent study (Phan et al., 2001) showed that several compounds including chalcones
isolated from Chromolaena odorata are powerful antioxidants, protecting cultured skin cells against oxidative damage. It has been shown that reactive oxygen and nitrogen species contribute to the pathophysiology of inflammatory conditions. A recent report indicates that a chalcone derivative inhibited chemiluminescence in vitro, cell migration and eicosanoid and TNF-alpha in mouse air pouch injected with zymosan. This chalcone derivative also exerted inflammatory effects in the carrageenan paw edema (Herencia et al., 2001). Hsieh et al. (1998) showed that 2′,5′-dihydroxychalcone may be a promising antiinflammatory agent,
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because it is a potent mediator and cyclooxygenase inhibitor. Their results indicated that the antiinflammatory effects of these compounds are mediated, at least partly, through the suppression of chemical mediators released from mast cells and neutrophils. Recently, Nakamura et al. (2002) synthesized fluorinated 3,4-dihydroxychalcones which were shown to be 5-lipoxygenase inhibitors and inhibitors of peroxidation in rat liver microsomes. Correa et al. (2001) synthesized eleven chalcones which were tested for their antinociceptive activity using the writhing test in mice. The authors showed that some of these compounds, after intraperitoneal administration, caused potent and dose-related antinociception. Among the compounds tested, 3,4-dichlorochalcone was the most effective one and, in the formalin test, inhibited only the inflammatory pain (second phase of the test). In the present work, we studied antinociceptive and antiedematogenic activities of a chalcone-enriched fraction (CEF) isolated from M. urundeuva, a medicinal plant largely used in Northeast Brazil for the treatment of several diseases, especially as an antiinflammatory in gynecological disorders. We showed a significant and dose-dependent antinociceptive effect of chalcones at low doses, after intraperitoneal administration, in the writhing test in mice. These compounds were less effective orally, probably due to a somewhat erratic absorption. In the formalin test, which measures pain of both neurogenic (first phase) and inflammatory (second phase) origins, the CEF inhibited preferentially the second phase of the response, and the opioid system seems to have a role in this effect. A small but significant decrease in carrageenan-induced paw edema was also detected, after intraperitoneal as well as after oral administration of a higher dose. Antiinflammatory effects have already been demonstrated with chalcones (Hsieh et al., 1998), and some chalcone derivatives have been reported to be potent chemical mediators and cyclooxygenase inhibitors. Hsieh et al. (1998) showed that some chalcones present strong inhibitory effects on the release of β-glucuronidase and histamine from rat peritoneal mast cells stimulated with compound 48/80. Other chalcones exhibited potent inhibitory effects on the release of β-glucuronidase and lysozyme from rat neutrophils stimulated with fMLP. Also, Lin et al. (1997) tested some chalcone derivatives for their inhibitory effects on platelet aggregation and activation of mast cells and neutrophils. They reported that the arachidonic acid-induced platelet aggregation was inhibited potently by almost all those compounds, and some also showed a potent inhibitory effect on collagen-induced platelet aggregation and cyclooxygenase. The authors suggest
that the anti-platelet effects of chalcones are mainly a result of the inhibition of thromboxane formation. It is possible that at least part of the antiinflammatory effect observed in the present work may also be due to platelet anti-aggregation, as we observed that CEF inhibited around 20% of ADP-induced-aggregation in human platelets (data not shown). Analgesic and anti-inflammatory effects have already been observed in flavonoids as well as in tannins (Ahmadiani et al., 1998; Ahmadiani et al., 2000). In the present work, the observed CEF antinociceptive effect is probably related to the opioid system. Although antiinflammatory effects have also been detected in chalcones, the present results are, as far as we know, the first report of peripheral and central analgesic as well as antiinflammatory effects of dimeric chalcones from Myracrodruon urundeuva. Acknowledgments
Authors are grateful to the technical assistance of Ms. M. Vilani Rodrigues Bastos. The work had the financial support of the Brazilian National Research Council (CNPq) and the Research Support Foundation (FUNCAP) of the State of Ceará, Brazil.
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