Chemical Constituents and Analgesic Activity of Conocliniopsis prasiifolia.

ABSTRACT

The phytochemical study of Conocliniopsis prasiifolia. (DC) R. M. King & H. Robinson (Asteraceae) yielded four substances which were identified by spectral analysis as the flavonoids 5-hydroxy-4′,7-dimethoxyflavone, 4′,5,6,7-tetramethoxyflavone, 4′,5,7-trimethoxyflavone and 4′,5-dihydroxy-7-methoxyflavone, described here for the first time in this species. The evaluation of analgesic activity of the crude extract, carried out by the hot plate test in mice, suggests a potent antinociceptive effect.

Keywords:

• Conocliniopsis prasiifolia
• Asteraceae
• flavonoids
• analgesical activity

Introduction

Conocliniopsis prasiifolia. (DC) R. M. King & H. Robinson belongs to the family Asteraceae, which consists of 1100 genera and 25000 species with a large geographic distribution. In Brazil the family is represented by approximately 180 genera (Barroso, 1986). The plant, known in folk medicine as “mentrasto” is popularly used as an anti-inflammatory, analgesic and antipyretic (Pio-Correia, 1984). Previous studies with this species described the presence of two classes of natural compounds, the heliangolides (Bohlmann et al., 1980) and bejaranolides (Bohlmann et al., 1984). The present work describes for the first time the occurrence of flavonoids in C. prasiifolia. as well as the analgesic activity of the crude extract.

Materials and Methods

General experimental procedures

¹H and ¹³C NMR spectra were measured at 200 MHz for ¹H and 50 MHz for ¹³C, using CDCl₃ as solvent.

Plant material

The sample of Conocliniopsis prasiifolia. was identified by Prof. Maria de Fátima Agra. The aerial parts were collected in October 2001, near the city of Santa Rita, state of Paraíba, Northeastern Brazil. A voucher specimen (AGRA 2052) is deposited at the Herbarium Lauro Pires Xavier (JPB) of the Universidade Federal da Paraíba.

Extraction and isolation

The dried and ground aerial parts (3.8 kg) were exhaustively extracted with 95% EtOH at room temperature. The extractive solution was concentrated under vacuum yielding 338 g of the crude extract. This was fractionated with ethyl ether, yielding the ethereal phase and a insoluble portion. The ethereal phase was suspended in a solution of MeOH:H₂O (3:2) and partitioned with hexane and CHCl₃. The chloroform fraction was subjected to column chromatography over silica gel eluting with hexane, CHCl₃ and MeOH. The fractions were monitored by TLC, yielding, after purification, four compounds:

5′-hydroxy-4.′,7-dimethoxyflavone. (52 mg): ¹H NMR (CDCl₃ δ): 3.86 (s., MeO-4′), 3.86 (s., MeO-7), 6.34 (d., J. = 1.2, H-6), 6.56 (s., H-3), 6.99 (d., J. = 8.8 Hz, H-2′ and H-6′), 7.81 (d., J. = 8.6 Hz, H-3′ and H-5′). ¹³C NMR (CDCL₃ δ): 55.3 (MeO-4), 55.8 (MeO-7), 92.6 (C-8), 98.0 (C-6), 104.2 (C-3), 105.5 (C-10), 114.4 (C-3), 114.4 (C-5), 123.5 (C-1), 128.0 (C-2), 128.0 (C-6′), 157.6 (C-5), 162.1 (C-9), 162.5 (C-2), 163.4 (C-4), 165.3 (C-7), 182.3 (C-4).

4.′,5.,6.,7.-Tetramethoxyflavone. (23 mg): ¹H NMR (CDCl₃ δ): 3.83 (s., MeO-4′), 3.87 (s., MeO-5), 3.94 (s., MeO-6), 3.94 (s., MeO-7), 6.56 (s., H-3), 6.76 (s., H-8), 6.95 (d., J. = 9.0 Hz, H-2′ and H-6′), 7.78 (d., J. = 8.8 Hz, H-3′ and H-5′). ¹³C NMR (CDCl₃ δ): 55.4 (MeO-4′), 56.2 (MeO-5), 61.5 (MeO-6), 62.1 (MeO-7), 96.2 (C-8), 106.7 (C-3), 112.5 (C-10), 114.3 (C-3′), 114.3 (C-5′), 123.6 (C-1′), 127.5 (C-2′), 127.5 (C-6′), 140.2 (C-6), 152.3 (C-5), 155.0 (C-9), 157.5 (C-7), 161.1 (C-4′), 162.0, (C-2), 177.0 (C-4).

4′,5,7-Trimethoxyflavone. (42 mg): ¹H NMR (CDCl₃ δ): 3.81 (s., MeO-4′), 3.85 (s., MeO-5), 3.89 (s., MeO-7), 6.29 (d., J. = 1.8 Hz, H-8), 6.48 (d., J. = 1.8 Hz, H-6), 6.55 (s., H-3), 6.92 (d., J. = 8.4 Hz, H-2′ and H-6′), 7.74 (d., J. = 8.4 Hz, H-3′ and H-5′). ¹³C NMR (CDCl₃ δ): 55.4 (MeO-4′), 55.7 (MeO-7), 56.3 (MeO-5), 92.6 (C-8), 95.9 (C-6), 107.2 (C-3), 108.0 (C-10), 114.2 (C-3′), 114.2 (C-5′), 123.5 (C-1′), 127.4 (C-2′), 127.4 (C-6′), 159.6 (C-5), 159.6 (C-4′), 160.6 (C-9), 161.9 (C-2), 163.8 (C-7), 177.4 (C-4).

4′,5-Dihydroxy.-7-methoxyflavone. (48 mg): ¹H NMR (CDCl₃ δ): 3.76 (s., MeO-7), 6.61 (d., J. = 2.4 Hz, H-8), 6.70 (d., J. = 2.4 Hz, H-6), 6.93 (s., H-3), 7.26 (d., J. = 8.8 Hz, H-2′ and H-6′), 7.94 (d., J. = 8.8 Hz, H-3′ and H-5′). ¹³C NMR (CDCl₃ δ): 56.0 (MeO-7), 92.9 (C-8), 98.6 (C-6), 104.1 (C-3), 105.9 (C-10), 116.9 (C-3′), 116.9 (C-5′), 122.1 (C-1′), 129.0 (C-2′), 129.0 (C-6′), 158.1 (C-5), 162.7 (C-4′), 162.8 (C-9), 164.8 (C-2), 165.8 (C-7), 182.8 (C-4).

Study animals

Male Swiss mice (28-33 g) were used throughout this study. The animals were randomly housed in appropriate cages at 25 ± 2°C on a 12 h light/dark cycle (lights on 6:00-18:00) with free access to food (Purina) and water. They were used in groups of ten animals each. Experimental protocols and procedures were approved by the Laboratório de Tecnologia Farmacêutica Animal Care and Use Committee.

Hot-plate test

The hot-plate test was used to measure response latencies according to the method described by Eddy and Leimback (1953). Mice were preselected on the hot plate at 50 ± 3°C. Animals were then treated with EHCp 100 mg/kg (i.p.) and 500 mg/kg (p.o.), vehicle (Tween 80 0.2%) and morphine 5 mg/kg (i.p.). The reaction time(s) for each mouse was determined on the hot-plate test before and after drug administration at intervals of 30 min for a total period of 120 min. To avoid possible injury, a cut-off period of 35 sec was followed while measuring the reaction time.

Statistical analysis

The data are expressed as mean±S.E.M. And the statistical significance was determined using an analysis of variance (ANOVA) followed by Student's t.-test. Values were considered significantly different at p < 0.05.

Results and Discussion

The EHCp 100 mg/kg (i.p.) and 500 mg/kg p.o. shows a significant antinociceptive effect with the hot-plate test. The hot-plate thermal stimulation was used to evaluate the analgesic activity mediated by central mechanisms (Al-Ghamdi, 2001). However, the reaction time for both groups treated with the EHCp (100 mg/kg i.p. and 500 mg/kg p.o.) significantly greater than that the control (p < 0.05) indicates central analgesic effect, similarly occurs with morphine (5 mg/kg i.p.) (Table 1). Results suggest that EHCp possesses a potent antinociceptive effect. In addition, the analgesic effect of Conocliniopsis prasiifolia. presented in this study is consistent with used traditional medicine (Matos, 1998; Diniz et al., 1998), the mechanism by which it occurs is not fully understood. In conclusion, the results suggest that the hydroalcohol extract of the leaves of Conocliniopsis prasiifolia. possess an analgesic potential that supports the folk medicinal use of this plant. However, further studies currently in progress will enable us to understand the mechanism of action underlying the effects observed in this investigation.

Table 1 Effect of the inhibition of EHCp on hot plate test in mice.

Reaction Time±EPM
Group	Dose	Administration via	No. of animals	0 min	30 min	60 min	90 min	120 min
Control	Tween 80 0.2%	i. p.	8	13.3	13.4	13.4	13.7	14.0
EHCp	100 mg/kg	i. p.	8	13.9	16.0	19.6	18.0	17.4
EHCp	500 mg/kg	v. o.	8	14.4	16.9	23.0	21.0	15.4
Morphine	5 mg/kg	i. p.	8	13.9	24.9	26.8	23.2	24.9

Analysis of variance test (ANOVA),

Table 1 shows that EHCp (100 mg/kg i.p. and 500 mg/kg p.o.) significantly increased (p < 0.05) the reaction time as compared to the control group (treatment with vehicle) in the times of 60 and 90 min after administration. In the morphine treated animals, similarly occurs a significantly increased (p < 0.05 and p < 0.01) reaction times.

Acknowledgments

The authors are grateful to Prof. Maria de Fátima Agra for botanical identification, J. C. Duarte for technical assistance and to the Brazilian agencies CNPq and CAPES for financial support.

Notes

*p < 0.05, n = 3, test experiment.