Journal of Ethnopharmacology 105 (2006) 148–153
Antinociceptive effect from Ipomoea cairica extract A.A. Ferreira a , F.A. Amaral b , I.D.G. Duarte b , P.M. Oliveira a , R.B. Alves a , D. Silveira c , A.O. Azevedo b , D.S. Raslan a , M.S.A. Castro b,∗ a
Departamento de Qu´ımica, Instituto de Ciˆencias Exatas, Universidade Federal de Minas Gerais, Minas Gerais, Brazil b Departamento de Farmacologia, Instituto de Ciˆ encias Biol´ogicas, Universidade Federal de Minas Gerais, Av. Antˆonio Carlos 6627 Pampulha, Belo Horizonte, 31270-901 Minas Gerais, Brazil c Faculdade de Ciˆ encias da Sa´ude, Universidade de Bras´ılia, Bras´ılia-DF, Brazil Received 8 November 2004; received in revised form 5 October 2005; accepted 13 October 2005 Available online 22 November 2005
Abstract Ipomoea cairica L. Sweet (Convolvulaceae) is used in Brazilian folk medicine for the treatment of rheumatism and inflammations. Ipomoea cairica ethanolic extract (100, 300, 1000 and 3000 mg/kg; per os) induced dose-dependent reduction of response in the formalin test inflammatory phase in mice. The same dose range did not modify neurogenic pain in formalin test, tail-flick reflex latency, carrageenan-induced paw edema, and Rota-Rod test motor performance. From the bio-active fraction 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid were obtained. These compounds have been previously reported to have analgesic and antioxidative effects. A possible explanation for the antinociception is that somehow the compounds present in the extract reduced the release of pro-nociceptive mediators unrelated to carrageenan-induced edema, such as histamine. Interestingly, caffeoylquinic acid derivatives have been reported to inhibit histamine release on in vitro models. The isolated caffeoylquinic acids could explain, at least in part, the antinociceptive effect of Ipomoea cairica polar extract. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Ipomoea cairica; Convolvulaceae; Antinociceptive; Anti-inflammatory; Di-O-caffeoylquinic acids
1. Introduction Ipomoea cairica L. Sweet (Syn. Ipomoea turberculata (Desr.) Roem. & Schult.) (Convolvulaceae) is a climbing plant widely distributed around almost all tropical regions and is used in folk medicine all over the world. In Brazil, this species is usually found in grazelands that were originally vegetative. Cachac¸a, a typical Brazilian alcoholic drink obtained by the fermentation and later distillation of sugar cane sap, is used to macerate of the aerial parts. The macerate is frequently used orally as an anti-inflammatory, anti-rheumatic, for treatment of syphillis and diarrhea (Pio Correa, 1978; Franco and Fontana, 1997). Accord-
Abbreviations: ACF, acetonitrile–chloroform fraction; AqF, aqueous fraction; CG, carrageenan; DAD, photodiode-array detector; EtOH-Ipc, ethanolic extract of Ipomoea cairica; HPLC, high-performance liquid chromatography; HxF, hexane fraction; IASP, International Association for the Study of Pain; i.pl., intraplantar; P, mean pain; PBS, phosphate-buffered saline; S.E.M., standard error medium; TLC, thin layer chromatography; TMS, tetramethylsilane ∗ Corresponding author. Tel.: +55 21 31 34992711. E-mail address:
[email protected] (M.S.A. Castro). 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.10.012
ing to Thomas et al. (2004), a drink made from crushed leaves is used for the treatment of body rashes especially those accompanied by fever. Although Ipomoea cairica is widely used in folk medicine, few data about its biological activities are found in literature. On the other hand, some isolated compounds have been reported to be effective when tested for biological activities. Chemical studies of Ipomoea cairica led to isolation of arctiin, arctigenin, trachelogenin, matairesinol, umbeliferone, -sitosterol, scopoletin, fatty acids (Nishibe et al., 1993; Lima and Braz-Filho, 1997), 4 ,7-dimethylquercetin, 7-O-glucopyranosil-4 -methylapigenin, friedelinol (Lin and Chou, 1997) and cyanidin-3-(p-coumaryl-caffeoyl)-sophoroside (Pomilio and Sproviero, 1972). The essential oil of this species presented larvicidal properties against larvae of Culex tritaeniorhynchus, Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus (Thomas et al., 2004). The lignans (−)-arctigenin and (−)-trachelogenin were found to inhibit replication of human immunodeficiency virus type I (HIV-1; strain HTLV-III B) in vitro (Schroder et al., 1990) and significant anti-tumor and Ca2+ -antagonist activities (P`aska et al., 1999). In addition, arctigenin showed significant
A.A. Ferreira et al. / Journal of Ethnopharmacology 105 (2006) 148–153
neuroprotective activity against glutamate-induced toxicity in primary-cultured rat cortical cells by inhibition of the activation of MAP kinases, including ERK1/2, p38 kinase and JNK through the inhibition of MKK activities (Jang et al., 2002; Cho et al., 2004). The acetic acid-induced writhing test showed that -sitosterol decreased the number of squirms induced by acetic acid and the hot plate method confirmed its analgesic activity. -Sitosterol also exhibited anthelminthic and antimutagenic activities (Villasenor et al., 2002). Considering the frequent indication of Ipomoea cairica for the treatment of inflammations of several etiologies in Brazilian folk medicine, our group carried out a phytochemical bio-guided survey by in vivo antinociceptive tests. 2. Materials and methods 2.1. Plant and material Aerial parts from Ipomoea cairica were collected at Governador Valadares, MG, Brazil, in May 2000 and identified by Beatriz G. Brasileiro. A voucher specimen was deposited at the Herbarium of the Natural History Museum/UFMG, Brazil (No. BHCB 16525). 2.2. Preparation of the extract and fractions The air-dried powdered plant material (3.3 kg) was extracted at room temperature with ethanol for 7 days (repeated three times). After filtration, ethanol was removed under reduced pressure, which furnished 250 g (7.6%) dry extract. The crude ethanolic extract of Ipomoea cairica (EtOH-Ipc, 20.0 g) was partitioned with n-hexane–CH3 CN–CHCl3 –H2 O (2:3.4:1:1) and provided furnished three fractions: n-hexane (HxF, 5.5 g, 27.5%), CH3 CN–CHCl3 (ACF, 8.5 g, 42.5%) and aqueous fraction (AqF, 5.8 g, 29.0%). Crude extract and fractions were used in pharmacological assays. The same partition procedure was repeated to obtain an additional amount of AqF for phytochemical study.
149
The purity of the substance was confirmed by HPLC analysis. Identification of these compounds was performed through NMR analyses (1 H NMR, 13 C NMR, COSY, HSQC and HMBC), IR and UV spectral data. The data obtained were compared with values found in literature. 2.4. Instrumentation HPLC analysis was carried out on a Shimadzu ClassVP equipment with reversed-phase column Shim-pack (Shimadzu, Kyoto, Japan; 250 mm × 4.6 mm) eluted with 0.01% H3 PO4 –CH3 CN (68:32) at a constant flow rate of 1 mL/min. Constituents were detected at 300 nm using a DAD (SPDM10AVP) detector. IR spectra were measured in KBr on a Shimadzu/IR-408 spectrophotometer. 1D (1 H and 13 C) and 2D NMR (COSY, HSQC and HMBC) spectra were recorded in CD3 OD solution on a Bruker DRX 400 Avance NMR spectrometer operating at 400 and 100 MHz for 1 H and 13 C, respectively. TMS was used as an internal proton standard and the solvent signal (δCD3 OD 49.0 ppm) as an internal carbon standard. X-ray diffraction analysis was carried out on a Rigaku GEIGERFLEX diffractometer. Silica gel 60 (70–230 mesh, Merck) was used for column chromatography and silica gel 60 F254 plates (Merck) were used for TLC. 2.5. Animals Male Swiss mice (25–35 g, CEBIO/UFMG, Brazil) were housed in plastic cages with free access to food and water, under a 12-h light:12-h dark cycle (lights on at 07:00 a.m.). Experiments were conducted between 09:00 a.m. and 05:00 p.m., on previously fasted (12–18 h) animals. All testing procedures were in accordance with the ethical guidelines of the IASP (Zimmermann, 1983). 2.6. Formalin-induced nociception
2.3. Isolation of constituents Dried AqF (37.3 g) was suspended in EtOH and the insoluble portion was filtered. The residue was crystallized from MeOH to afford KCl (4.6 g, 12.33%). The EtOH-soluble portion from AqF was chromatographed on silica gel column by using an increasing gradient polarity starting with CH2 Cl2 –MeOH–H2 O (80:20:1) up to CH2 Cl2 –MeOH–H2 O (10:90:12). Fractions of 200 mL were collected. This procedure yielded 100 fractions, which were monitored by TLC. Fractions eluted with CH2 Cl2 –MeOH–H2 O (45:55:8) were pooled, dried and chromatographed on modified silica gel 60A (phosphate buffer) (Balis and Payne, 1971) eluted with n-hexane–EtOAc (up to 100% EtOAc) and EtOAc–MeOH (up to 10% MeOH), affording 87 fractions of 10 mL each. Eluates with similar TLC profiles were pooled to give seven combined fractions. This procedure yielded compounds 1 (22.5 mg, 0.06%) and 2 (90.2 mg, 0.24%) in pools 2 and 4, respectively.
This test was based on the method of Dubuisson and Dennis (1977) adapted for mice by Hunskaar and Fasmer (1985). Briefly, formalin solution (2% in PBS; 30 L/paw) was injected into the hind paw plantar surface (i.pl. injection). The time (s) spent licking or biting the affected paw was rated during two time intervals: 0–5 min (first phase or neurogenic pain) and 15–30 min (second phase or inflammatory pain). Oral treatment (p.o.) with sodium diclofenac (10 mg/kg; positive control), morphine sulphate (5 mg/kg; positive control), vehicle (water, 10 mL/kg), Ipomoea cairica ethanolic extract (0.1–3 g/kg), its fractions (HxF, 140 mg/kg; ACF, 213 mg/kg; AqF, 150 mg/kg) or KCl (150 mg/kg) were given 60 min prior to formalin injection. In order to estimate the doses of the HxF, ACF, AqF and KCl, we first determined the yield of each one relative to the EtOH-Ipc extract. The target doses used were threefold the reference doses (RD), which were based on the yield of each fraction and EtOH-Ipc extract ID50 . The reference doses were calculated
150
A.A. Ferreira et al. / Journal of Ethnopharmacology 105 (2006) 148–153
as follows: RD =
ID50 × YF 100
where ID50 is the inhibitory dose of EtOH-Ipc and YF is the yield of the fraction. 2.7. Tail-flick nociceptive reflex A constant focused heat was delivered to the mice’s tail, according to a previously described procedure (D’Amour and Smith, 1941). The tail-flick reflex latency (s) was measured every 30 min, from 1 h before (basal values) up to 2 h after oral treatment with vehicle (water, 10 mL/kg), morphine sulphate (5 mg/kg; positive control), EtOH-Ipc or its fractions (as described above). The animals whose basal responses were >6 s were discarded and a cut-off time of 9 s was maintained throughout the experiment.
2.9. Motor performance test (Rota-Rod) Motor performance was measured as time (s) spent walking on a rotating rod (16 rpm) during 2-min trials (Rota-Rod, Ugo Basile mod. 7600). Mice were submitted to three training sessions 24 h before testing and those scoring
