August 2005 Chem. Pharm. Bull. 53(8) 881—885 (2005) 881 Evaluation of Strychnos pseudoquina ST. HIL. Leaves Extract on Gastrointestinal Activity in Mice Marcelo Aparecido da SILVA,a,b Bruna Paola Murino RAFACHO,c Clelia Akiko HIRUMA-LIMA,c Lúcia Regina Machado da ROCHA,c Lourdes Campaner dos SANTOS,a Miriam SANNOMIYA,a Alba Regina Monteiro SOUZA-BRITO,d and Wagner VILEGAS*,a a Departamento de Química Orgânica; Instituto de Química; c.p. 355, CEP 14800–900, UNESP, Araraquara, SP, Brasil: Departamento de Fármacos e Medicamentos, Faculdade de Ciências Farmacêuticas; CEP 14800–900, UNESP, Araraquara, SP, Brasil: c Departamento de Fisiologia, Instituto de Biociências; c.p. 510, CEP 18618–000 UNESP, Botucatu, SP, Brasil: and d Departamento de Fisiologia e Biofísica, Instituto de Biologia; c.p. 6109, CEP 13083–970, UNICAMP, Campinas, SP, Brasil. Received November 1, 2004; accepted March 25, 2005 b Strychnos pseudoquina ST. HIL. (Loganiaceae) was investigated for its ability to protect the gastric mucosa against injuries caused by non-steroidal anti-inflammatory drugs (piroxicam) and a necrotizing agent (HCl/EtOH) in mice. The MeOH extract and enriched alkaloidic fraction (EAF) provided significant protection in experimental models wheer used at doses of 250 and 1000 mg/kg. In vivo tests were carried out to evaluate for possible toxic effects and no mortality was observed up to the 5 g/kg dose level. Phytochemical investigation led to the isolation of a new indole alkaloid, which elucidated the observed pharmacological effects. Key words Strychnos pseudoquina; antiulcer activity; toxic activity; alkaloid As part of a state collaborative program named BIOTAFapesp, our project entitled “Sustainable use of the Brazilian Biodiversity: Chemical and Pharmacological Prospection on Higher Plants” aims to investigate plants with potential activity on the prevention of gastric injuries. This research is based on ethnopharmacological investigation, followed by the chemical and pharmacological investigation of plant extracts. In this work, we investigated the leaves of Strychnos pseudoquina ST. HIL. (Loganiaceae). Strychnos pseudoquina is used in traditional medicine for the treatment of malaria and for the preparation of so-called “aqua inglesa,” largely used in Brazil as a tonic and febrifuge and also to relieve stomach pains.1) It is popularly known as “quina branca” (white quine). Many plants from the genus Strychnos are also called “quina,” since they are used against malaria.2) Phytochemical investigations of several Strychnos species led to the isolation of a number of indole alkaloids as the main compounds. Flavonoids, terpenoids and waxes were also reported to a lesser extent. Previous investigation of S. pseudoquina afforded the alkaloids bisnordihydroxytoxiferine, diaboline and 11-methoxydiaboline, as well as the flavonoids isorhamnetine and estricnobiflavone.1,3) Despite the well known toxicity of many Strychnos species, our ethnopharmacological survey indicated that S. pseudoquina is used as a bitter folk medicine against stomach diseases. Since Strychnos species are known to contain toxic compounds, it is important to conduct a scientific investigation in order to assure its efficacy and/or to detect the presence of toxic components.4) Results and Discussion For this phytochemical examination we used an aliquot of the methanolic extract of leaves from S. pseudoquina. TLC plates sprayed with Draggendorff, iodoplatinate, and NP/ PEG (Natural Products/Polyethyleneglycol) reagents indicated that this polar extract contained alkaloids and flavonoids.1,3) This extract was then fractionated by gel permeation chromatography over Sephadex LH-20, and the frac- tions were purified by adsorption chromatography on silica or alumina, affording compounds 1—3, which were identified by several spectroscopic methods. After analyses, 2 and 3 were identified as rutin and kempferol 3-O-b -rutinoside. Compound 1 was identified as a pale yellow gum. The IR spectrum presented bands at 3300 cm⫺1 (OH) and 1600 cm⫺1 (C⫽C). The molecular formula of 1 was determined to be C19H22N2O2 by the EI-MS, which showed pseudo molecular ion peak sat m/z 311 [M⫹H]⫹ and m/z 333 [M⫹Na]⫹, and the elemental analysis data. The structure of 1 was fully elucidated by 1D and 2D NMR experiments at 500 MHz. The 1 H-NMR spectrum in CDCl3 (Table 1) displayed signals at d 7.05 (d, J⫽8.0 Hz), 6.71 (dd, J⫽8.0, 8.0 Hz), 7.00 (dd, J⫽8.0, 8.0 Hz) and 6.57 (d, J⫽8.0 Hz), corresponding to an ortho-dissubstituted benzene ring. These signals and the signal at d 4.03 suggested the presence of an indolic nucleus.5—7) The signal at d 6.16 (d, J⫽1.5 Hz) suggested the presence of a double bond in the structure of 1. The ⬎C⫽CH–CH3 system was deduced from the signal at d 5.54 (q, J⫽6.5 Hz, ⫽CH–) coupled to the doublet at d 1.67 (J⫽6.5 Hz, –CH3) in the 1H–1H COSY (gradient correlate spectroscopy) spectrum. Other signals corresponding to the aliphatic chain of 1 were observed between d 1.97 and 4.03 (Table 1). The 13C-NMR spectrum presented 19 signals, eight of which could be assigned to the indolic nucleus: d 54.5 (C-7), 65.0 (C-2), 110.6 (C-12), 119.9 (C-10), 122.5 (C-9), 128.8 (C-11), 132.4 (C-8) and 146.3 (C-13). The signals at d 139.9 (C-16), 124.4 (C17), 124.1 (C-19) and 130.2 (C-20) confirmed the presence of two double bonds. The presence of the signal at d 91.8 indicated the presence of an additional OH group at position C-3.6,7) The shielding effects on C-5, C-6 and C-21 when compared to the literature also support the presence of an OH-group at C-3.6,7) When compared to the literature, the deshielding a -effect over C-17 and b -effect over C-16 suggest the presence of an OH-group at C-17.6,7) The structure of 1 was deduced from a combination of 1D NOESY (nuclear overhauser effect spectroscopy), 1H–1H- ∗ To whom correspondence should be addressed. e-mail: vilegasw@iq.unesp.br. © 2005 Pharmaceutical Society of Japan 882 Vol. 53, No. 8 Table 1. 1 H- and 13C-NMR Data of 1 (CDCl3, 500 MHz) Position 1 H (JHH in Hz) 13 C COSY gHMQC gHMBC 2 3 4.03 (s) — 65.0 91.8 H2/H17 — H2/C2 — 5 H5a 2.88 (m); H5b 3.72 (m) 52.4 6 2.21 (m) 39.0 H5a/C5 H5b/C5 H6/C6 7 8 — — 54.5 132.4 H5a/H5b H5b/H5a H6/H5a H6/H5b — — 9 10 7.05 (d, J⫽8.0) 6.71 (dd, J⫽8.0, 8.0) 122.5 119.9 H9/C9 H10/C10 11 7.00 (dd, J⫽8.0, 8.0) 128.8 H11/C11 C11/H9 12 13 6.57 (d, J⫽8.0) — 110.6 146.3 H9/H10 H10/H11 H10/H9 H11/H12 H11/H10 H12/H11 — — C8/H10 C8/H12 C9/H11 C10/H12 H12/C12 — 14 H14a 2.03 (d, J⫽14.5) H14b 1.97 (dd, J⫽14.5, 9.0) 32.2 C12/H10 C13/H11 C13/H9 — 15 3.43 (d, J⫽9.0) 31.4 16 17 — 6.16 (d, J⫽1.5) 18 19 — — H14a/H14b H14b/H14a H14b/H15 H14a/H15 H15/H14b H15/H14a H14a/C14 H14b/C14 139.6 124.4 — H17/H2 — H17/C17 1.67 (dd, J⫽6.0, 1.0) 5.54 (q, J⫽6.0) 13.6 124.1 H18/H19 H19/H18 H18/C18 H19/C19 20 — 130.2 — — 21 H21a 3.80 (m) H21b 3.42 (m) 54.1 H21a/H21b H21b/H21a H21b/H18 H21a/C21 H21b/C21 COSY, gHMQC (gradient heteronuclear through multiple quantum coherence) and gHMBC (gradient heteronuclear multiple bond correlations) experiments, as presented in Fig. 1. Correlation in the HMBC spectrum between the proton at d 3.80 (H21a) and the carbon at d 91.8 (C-3) supported the presence of an additional OH-group at C-3, whereas the long-range correlation between the proton at d 6.16 (H17) and the carbons at d 139.6 (C-16) and at d 65.0 (C-2) agree with an OH-group at position C-17. The structure of 1 is the 3-hydroxy-enolate of the known alkaloid nordiidrofluorocurarine, previously isolated from S. amazonica and S. froessi.8) We can pose the question of whetter compound 1 has a role as a precursor in the biosynthesis of strychnine,9) since 1 seems to be originated just before the formation of the Wieland–Gumlich aldehyde (Fig. 2), but without hydroxylation at positions 3 and 18. It was already reported that hydroxylation at position 3 can result from an artifact produced when chlorinated solvents are used in the extractive processes.10) To check this possibility, we extracted the leaves of S. pseudoquina with pure MeOH and with water. TLC analyses using the isolated substance 1 as standard showed the presence of 1 in both extracts, thus suggesting that 1 is not an artifact. Although much work has been done on the pharmacological properties and chemical composition of species belong- Fig. 1. quina H15/C15 C2/H17 C3/H21a C3/H17 — — C15/H19 C15/H17 C15/H18 C15/H2 C16/H2 C17/H16 C17/H2 — C19/H18 C19/H21b C20/H18 C20/H21a C21/H19 Indole Alkaloid 1 Isolated from the Leaves of Strychnos pseudo- ing to the genus Strychnos, only limited data are available in the literature from the pharmacological and toxicity properties of S. pseudoquina that might ensure the safe use of this important medicinal plant. Due to the high cytotoxicity of some of the Strychnos alkaloids, we first evaluated the crude MeOH extract and the EAF in in vivo acute toxicity assays in mice. Animals were treated with a single dose of the MeOH extract and EAF (5000 mg/kg each) and observed during a period of 14 d. Results showed that there are no significant differences between the animals treated with saline and those treated with MeOH or EAF obtained from S. pseudoquina (Fig. 3). In agreement with this, no signs or symptoms of acute toxicity were observed. There were no significant differences in organ weight, in water or food intake, or in the amount of faeces August 2005 883 Fig. 2. Proposed Role of 1 on the Biogenetic Pathway to the Formation of the Wieland–Gumlich Aldehyde in the Biosynthesis of Strychnine (Based on Dewick, 1997) Table 3. Effects of the Lansoprazole, MeOH Extract and of the Enriched Alkaloidic Fraction (EAF) of S. pseudoquina in the Model of HCl/EtOH in Mice (Mean⫾S.D.) Dose (mg/kg) n pH (mean⫾S.D.) Saline Lansoprazole MeOH 10 30 250 1000 7 8 5 5 3.0⫾0.7 6.0⫾0.5** 2.4⫾0.5 2.8⫾1.1 47.0⫾27.6 5.7⫾3.2** 25.2⫾9.7 19.6⫾16.2* — 87.8 46.4 58.3 Saline Lansoprazole EAF 10 30 250 1000 8 8 5 5 2.2⫾0.5 5.5⫾1.3** 3.0⫾0.0 3.0⫾0.0 64.6⫾24.9 2.1⫾2.3** 27.2⫾4.3* 36.4⫾15.1* — 97.7 57.9 43.7 Treatments Fig. 3. Body Weight Gain in Mice Treated with Saline, MeOH Extract and Enriched Alkaloidic Fraction (EAF) of S. pseudoquina (5 g/kg, p.o.) during 14 d Table 2. Evaluation of the Acute Toxicity of MeOH Extract and Enriched Alkaloidic Fraction (EAF) of S. pseudoquina (5 g/kg, p.o.) in Mice (Mean⫾S.D.)a) Weight (g) (n⫽10) Control (10 ml/kg) MeOH (5 g/kg) Body Kidney Liver Heart Lungs Mortality 41.50⫾7.09 0.52⫾0.12 2.22⫾0.53 0.20⫾0.05 0.27⫾0.04 0/10 40.00⫾3.33 0.51⫾0.14 1.89⫾0.39 0.180⫾0.036 0.27⫾0.04 0/10 ANOVA F(2,27)⫽2.17. Dunnett test ∗ p⬍0.05. daily administration of MeOH extract and EAF. EAF (5 g/kg) 40.50⫾5.98 0.45⫾0.06 1.94⫾0.34 0.17⫾0.31 0.22⫾0.03 0/10 a) Results were obtained 4 d after produced by treated and control mice (Table 2). None of the treated mice died during the 14 d of observation after the administration of the MeOH extract or the EAF. These results add more information about the possible therapeutic safety of this species. The antiulcer activity of the crude MeOH extract and of ULI Inhibition (mean⫾S.D.) (%) ANOVA F(4,25) for MeOH⫽6.54 p⬍0.001. Test of Dunnett ∗ p⬍0.05; ∗∗ p⬍0.01; ANOVA F(4,25) for EAF⫽16.22 p⬍0.001. Test of Dunnett ∗ p⬍0.05; ∗∗ p⬍0.01. the EAF of S. pseudoquina was then evaluated by two different models. In the model of HCl/EtOH in mice (Table 3), oral administration of the crude MeOH extract (1000 mg/kg) significantly inhibited 58.3% of the ulcerogenic lesions from the HCl/ethanol solution over the gastric surface, whereas the EAF significantly inhibited 57.9 and 43.7% of the gastric ulcers at doses of 250 and 1000 mg/kg, respectively. NSAIDs (non-steroidal anti-inflammatory drugs), such as piroxicam, resulted in the production of gastric ulcers, mainly in the glandular portion of the stomach. Nearly 100% of the animals showed gastric ulceration. The majority of ulcers were gastric erosions that were multiple, dotted or elongated superficial haemorrhagic mucosal lesions that did not penetrate the muscularis mucosae.11) As shown in Table 4, in the NSAID-induced gastric ulceration model, pretreatment with crude MeOH extract produced a decrease in the gastric ulceration induced by piroxicam. The ulcerogenic lesions caused by piroxicam (Table 4) were significantly 884 Vol. 53, No. 8 Table 4. Effects of the Cimetidine, MeOH Extract and of the Enriched Alkaloidic Fraction (EAF) of S. pseudoquina in the Piroxicam Model in Mice (Mean⫾S.D.) Dose (mg/kg) n Saline Cimetidine MeOH 10 100 250 1000 7 5 5 5 2.6⫾0.5 3.0⫾0.0 2.6⫾0.5 2.8⫾0.4 21.1⫾6.2 6.0⫾1.6** 8.8⫾4.8** 6.4⫾4.4** — 68.0 55.5 63.2 Saline Cimetidine EAF 10 100 250 1000 8 8 5 5 3.0⫾0.5 3.4⫾0.5 3.0⫾0.0 3.0⫾0.0 31.4⫾9.3 10.0⫾4.8** 9.0⫾2.5** 8.0⫾3.4** — 68.1 71.2 74.5 Treatments pH ULI Inhibition (mean⫾S.D.) (mean⫾S.D.) (%) ANOVA F(4,22) for MeOH⫽7.11 p⬍0.001. Test of Dunnett ∗∗ p⬍0.01; ANOVA F(4,26) for EAF⫽20.70 p⬍0.001. Test of Dunnett ∗∗ p⬍0.01. inhibited by the crude MeOH extract, by 55.5% (250 mg/kg) and 63.2% (1000 mg/kg). The best results were obtained with the oral administration of EAF, with significant inhibition of the ulcerogenic lesions caused by piroxicam by 71.2% (250 mg/kg) and 74.5% (1000 mg/kg). These results show the effective antiulcer activity of EAF when compared with cimetidine treatment (68%), however no significant differences were observed between the groups treated with EAF and cimetidine (p⬎0.05). Therefore, our results show that MeOH extract and EAF from S. pseudoquina are orally effective against gastric damage induced by cytodestructive agents (HCl/ethanol) and ulcerogenic agents (NSAIDs). The mechanisms by which S. pseudoquina exerts its antiulcer activity is far from clear. However, EAF in particular is involved in the protection of the stomach against ulceration. It is interesting to observe that the structure of 1 roughly resembles that of omeprazole, a well known antiulcer agent. Literature reports that omeprazole can irreversibly bind to H⫹/K⫹ ATPase through the sulphydril group.12) However, the irreversible suppression of the gastric pump has been related to some carcinomas.13) In the case of 1, there is the possibility of a reversible bond between the alkaloid and the enzyme, thus revealing the promising potential of 1 as an anti-ulcer agent (Fig. 4). In conclusion, this study has shown that both the polar extract and the enriched alkaloid fraction of Strychnos pseudoquina have a significant anti-ulcer and cytoprotective effect on different experimentally induced gastric lesions, with as absence of toxicity by acute treatment. Our results show that the anti-ulcer activity of the MeOH extract is almost as potent as that of the alkaloid-enriched fraction. Therefore, it is possible that other compounds present in the crude extract (e.g. flavonoid glycosides) may also contribute to the observed activity. Despite the moderate potency of the extracts and fractions, the inhibition of gastric damage and absence of acute toxicity of S. pseudoquina show that the ethnopharmacological approach can be useful in the search for active extracts and compounds. Further studies are in progress to evaluate the chronic toxicity and the modes of action. Experimental Biological Material The leaves of Strychnos pseudoquina ST. HIL. were collected in May 2001, at Porto Nacional, Tocantins State, Brazil, and authenticated by Professor Eduardo Ribeiro dos Santos from Instituto de Biociências (IB), Universidade do Tocantins (UNITINS). A voucher speci- Fig. 4. Possible Mechanism of Action of 1 Showing the Reversibility of the Reaction men (Nr 3291) was deposited at the Herbarium of the UNITINS. Apparatus NMR spectra in CDCl3 were obtained using a Varian INOVA-500 spectrometer, operating at 500 MHz for 1H and at 125 MHz for 13 C and 2D-NMR (inverse detection 1H–13C HSQC and HMBC). ES-MS was performed on a Fisons VG Platform spectrometer in positive (70 V) mode. The sample was dissolved in MeOH and injected directly. IR spectrum was performed in a FT-IR-Nicolet Impact IMACT-400, KBr. UV spectra were obtained on a Beckman DU 670 spectrometer. Elemental analysis was made with a Carlo Erba EA 1110 apparatus. TLC was performed on silica gel SiF254 (Merck). The plates were visualized using UV light (254, 365 nm). Extraction and Isolation The leaves of S. pseudoquina (300 g) were powdered and successively extracted with CH2Cl2 and MeOH (1 week for each solvent). The solvents were evaporated in a vacuum to give 13.9 and 17.7 g of each extract, respectively. A portion (5.0 g) of the methylene chloride extract (CH2Cl2) was repeatedly fractionated by column chromatography on silica gel eluted with several gradient mixtures of hexane/EtOAc to give lupeol, a -amirin and b -amirin, identified by their NMR data compared to the literature,14) and also by GC-FID analyses with authentic standards. A portion (5.0 g) of the methanolic extract (MeOH) of S. pseudoquina was submitted to column chromatography on Sephadex LH-20 (100⫻5 cm) with MeOH as the eluent. One hundred fractions (5 ml) were collected and checked by TLC on silica gel plates CHCl3–MeOH–n-PrOH–H2O (5 : 6 : 1 : 4, v/v/v/v lower phase) was revealed with Draggendorff, iodoplatinate or NP/PEG (Natural Products/Polyethyleneglycol) reagents. Alkaloids were detected in Fr. 3—29 (named “enriched alkaloidic fraction (EAF)” ca. 3 g) and flavonoids were detected in Fr. 35—90 (250 mg). Fractions 23—25 (69.8 mg) were further purified by column chromatography on neutral alumina using CHCl3–MeOH–NH4OH (95.0 : 0.5 : 0.05, v/v/v) as an eluent to afford the alkaloid 1 (20 mg). Fractions 35—37 (40 mg) and fractions 40—42 (45 mg) were purified by repeated column chromatography on polyvinylpolypyrrolidone (Sigma) eluted with MeOH, yielding 2 (9.0 mg) and 3 (7.0 mg), respectively. Compounds 2 and 3 were identified by their NMR spectra and by comparison with previous data reported in the literature.15,16) Compound 1 IR (KBr): 3300 cm⫺1 (OH), 1600 cm⫺1 (C⫽C). ES-MS m/z (rel. int.) (70 V, positive ion): 311 (100) [M⫹H]⫹, 333 (30) [M⫹Na]⫹. Elemental analysis: Anal. C 73.45%, H 7.24%, N 9.15% Calcd for C19H22N2O2 C 73.52%, H 7.14%, N 9.03%. 1H- and 13C-NMR: see Table 1. Animals Male Swiss albino mice (25—35 g) from the Central Animal House of the Universidade Estadual Julio de Mesquita Filho (UNESP/Botucatu) were used. The animals were fed a certified Nuvilab CR-a® (Nuvital) August 2005 diet with free access to tap water under standard conditions of 12 h dark–12 h light and temperature (22⫾1%). Fasting was done prior to all assays because the standard drugs and extract were always administered orally (by gavage). Moreover, the animals were kept in cages with raised floors of wide wire mesh to prevent coprophagy. The protocols were approved by the UNESP Institutional Animal Care and Use Committee, following the recommendations of the Canadian Council on Animal Care.17) Drugs and Chemicals HCl, EtOH (Nuclear, Brazil), cimetidine (Sigma Chemical Co., St. Louis, MO, U.S.A.), piroxicam (Hexal, Brazil) and lansoprazole (Medley, Brazil) were used in this study. Each extract was dissolved in NaCl solution 0.9% (vehicle). All substances were prepared immediately before use, and the reagents used were of analytical grade. Hippocratic Screening and Acute Toxicity The signs and symptoms associated with the oral administration of the MeOH extract and of the EAF (5000 mg/kg, each) were monitored in 8 mice per dose. The mice were examined 0, 0.5, 1, 2, 4, 8, 24 and 48 h after administration to assess possible clinical or toxicological symptoms. The mortality rate was monitored for a period of 2 weeks.18) Antiulcerogenic Effect a) HCl/EtOH-Induced Ulcer: The antiulcerogenic activity of the extract obtained from S. pseudoquina was studied in HCl/EtOH-induced gastric ulcer. The experiment was performed as described in the literature.19) Mice were divided into groups of 7—8 animals which were fasted 24 h prior to receiving an oral dose of the vehicle, saline (10 ml/kg), lanzoprazole (30 mg/kg) or S. pseudoquina (250, 1000 mg/kg). After 50 min all groups were orally treated with 0.2 ml of a 0.3 M HCl/60% EtOH solution (HCl/EtOH) for gastric-ulcer induction. Animals were killed 1 h after the administration of HCl/EtOH, and the stomachs excised and inflated by saline injection (2 ml). The extent of the lesions was measured using an ulcerative lesion index (ULI), and the pH of gastric juice determined. This index was expressed as the sum of all lesions, as described in the literature.20) b) Non-steroidal Antiinflammatory Drug (NSAID)-Induced Gastric Ulcers in Mice: In this model,21) gastric ulcer was induced using piroxicam (30 mg/kg, s.c.) administered to mice. The crude MeOH extract or EAF from S. pseudoquina (250, 1000 mg/kg), cimetidine (100 mg/kg) or saline was administered orally 30 min before induction of the gastric ulcer. The animals were killed by cervical dislocation 4 h after treatment with the ulcerogenic agent; the stomachs were removed and the gastric juice pH and gastric damage were determined by the ULI calculated as described previously. Statistical Analysis Results ware expressed as the mean⫾S.D. Statistical significance was determined by one-way analysis of variance followed by Dunnett’s test, with the level of significance set at p⬍0.05. Acknowledgements We thank Fundação de Amparo à Pesquisa do Es- 885 tado de São Paulo (FAPESP) for a grant to D.R and M.S, the Biota-Fapesp Program for funding and Conselho Nacional de Desenvolvimento Científico Tecnológico (CNPq) for grants to W.V. and A.R.M.S.B. References 1) Nicoletti M., Goulart M. O. F., Delima R. A., Goulart A. E., DelleMonache F., Bettolo G. B. M., J. Nat. Prod., 47, 953—957 (1984). 2) Correa P. M., “Dicionário das Plantas Úteis do Brasil e das Exóticas Cultivadas,” 1st ed., Vol. VI, Imprensa Oficial, Rio de Janeiro, 1926. 3) Delle-Monache F., Aldo P. T., Bettolo G. B. M., Tetrahedron Lett., 25, 2009—2012 (1969). 4) Angenot L., Quertim-Leclercq J., Bisset N. G., J. Ethnopharmacol., 28, 1—52 (1990b). 5) Penelle J., Monique T., Philippe C., Viviane B., Michel F., Angenot L., J. Nat. 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