Abstract: Investigation of the twigs and leaves of the Ecuadorian medicinal plant Siparauna macrotepala belongs to the family Monimiaceae led to isolation and characterization of 7-hydroxy-1-oxo-14-norcalamenene, (+)-8-hydroxycalamenene, (+)-T-cadinol and 7,14-dihydroxycalamenene. The isolated compounds were identified by IR, NMR and MS spectroscopic analysis. The modern chromatographic technique SEPARO AB MPLC equipment was used for isolation of the compounds. The crude extracts showed low anti-inflammatory activity.
Introduction
Various Siparauna spp. (Monimiaceae) have been used in traditional medicine for treatment of headache, indigestion, rheumatism, snake bites, fever and rheumatism (Morton, 1981; Schultes and Raffauf, 1990) while extracts of various species of the genus have been used medicinally in the treatment of gastrointestinal disorders, skin diseases and modification of female sterility (Altschul, 1973) whereas some of these species are known to be poisonous to livestock (Blohm, 1962). The oxoaporphine alkaloids liriodenine and cassamedine have been isolated from S. guianensis (Braz-Fo et al., 1976; Gerard et al., 1986) while liriodenine and the oxoaporphine alkaloid oxonantenine have been reported as constituents of S. gilgiana (Chiu et al., 1982). S. macrotepala, was collected in 1992 as part of our project on chemical and pharmacological investigations of medical plants used in traditional medicine. In previous study (EI-Seedi et al., 1994) we reported the isolation and characterization of two new cadinane sesquiterpenes from S. macrotepala. In the present work I report the isolation and characterization of four sesquiterpenes for the first time from the species.
Materials and methods
General: The 1H and13C NMR spectra were recorded at 300 and 75 MHz respectively with TMS as an internal standard using a 300 MHz JEOL JNM Ex-270/4000 NMR instrument. The IR spectra were recorded with a Nicolet MX-S. The El-MS and FAB-MS (with glycerol as a matrix) spectra were recorded with a JEOL JMS SX/SX102A instrument.
The melting points were determined using a Digital Melting Point Apparatus (model IA 8103, Electrothermal Engineering Ltd, Southend-on-Sea, Essex, UK) and are uncorrected. TLC was performed on precoated aluminium sheets on silica 60 PF254+360, 0.25 mm (Merck, Darmstadt, Germany) and preparative TLC was performed on silica gel (60, PF254+360, Merck) glass plates, 20X20, 0.25, 0.5 and 1.8 mm (Merck). UV light (245 and 366 mm) and spraying with vanillin-sulphuric acid reagent followed by heating (120°, was used for detection.
Plant Material: Siparauna macrotepala is a small tree, 2-3 m, it consisting of small twigs and leaves and was collected at the road Loreto-Payamino, Provincia del Napo, Ecuador at an altitude of 500 m. A voucher specimen is deposited in the Herbario Economico, Escuela Politecnica Nacional, Quito, Ecuador.
Chromatography: Medium Pressure Liquid Chromatography (MPLC) was performed using a SEPARO AB MPLC equipment (Baeckstrom Separo AB, Lidingo, Sweden) (Jiron, 1996). SEPARO variable length glass columns with an inner diameter of 1.5 or 2.5 cm, packed with silica gel 60, 40-63 mm (Merck) were used. A FMI Lab pump, model QD (Fluid Metering Inc., Oyster Bay, NY, USA) was used at a flow rate of 20-30 mL/min. Fractions of 9 ml were collected with a Gilson fraction collector model 201. The columns were eluted with continuous gradients running from hexane, over CH2Cl2 to Me0H and H2O afforded by a SEPARO constant-volume mixing chamber combined with an open reservoir. Initially, the mixing chamber contained 50 mL non-polar solvent and the reservoir the first of 15-20 premixed binary (less polar/more polar solvent) gradient mixtures, of 20-40 mL each, which were successively fed to the reservoir during the separation.
Pharmacological study: Cyclooxygenase (COX) inhibiting activity assay: The COX-1 and COX-2 catalysed prostaglandin inhibiting capacities of extracts and compounds were performed according to literature (Noreen, 1998a).
Isolation and purification: The plant material consisting of twigs and leaves were dried at 40°C in the dark in a ventilated hood and grounded. The material, 1 kg, was exhaustively extracted at room temperature with occasional stirring with light petroleum (40-60°) and then with ethanol three times for 8 days each time. Evaporation of the solvents in vacua gave 24.4 g and 43.4 g respectively of crude oily extracts. The ethanol extract (43 g) was extracted with mechanical stirring at room temperature with ethyl acetate three times, 7 h for each crop; decantation of the solvent and evaporation in vacua at 45°C to gave 39.6 g. The insoluble part consists mainly of carbohydrates.
| Table 1: | 1H NMR spectral data of compounds 1-4 (MHz, CDCI3, 5 values) |
| *Not properly resolved | |
| Table 2: | 13C NMR spectral data of compounds 1-4 (MHz, CDCI3, 6 values) |
The ethyl acetate fraction, 28 g, was subjected to SEPARO AB MPLC on silica gel 60 (56 g) with gradient elution using hexane-methylene chloride and methylene chloride-methanol.
Compound 1, was purified by prep. TLC using petroleum ether: EtQAc (95:5) as eluent, 12 mg, oil. cm–1 3410, 1672, 1465, 1375 and 1365. 1H NMR and 13C NMR (Table 1, 2).
Compound 2, was purified by prep. TLC using petroleum ether: EtOAc (94:6) as eluent, 4 mg, oil. +37°(CHCI3; c = 1.2). cm–1 3390, 1620, 1460 and 1385. 1H NMR and 13C NMR (Table 1, 2).
Compound 3, was purified by silica gel column using petroleum ether:Et0Ac (90:10 and 80:20) as eluent, 11 mg, amorphous solid, mp 56-58°C, + 3.6° (CHCI3; c = 1.2). 1H NMR and 73C NMR (Table 1, 2).
Compound 4, was separated and purified by prep. TLC using petroleum ether:EtOAc (95:5) as eluent, 7 mg, colourless oil, cm–1 3360, 1618 and 1510. 11-1 NMR and 13C NMR (Table 1, 2).
Results
The crude petroleum ether and ethyl acetate extract showed low inhibition of cyclooxygenase (COX-1 and -2) catalysed prostaglandin (PG) synthesis in vitro (36 and 18% inhibition, 0.3 mg/mL) and (25 and 37% inhibition, 0.3 mg/mL) respectively (Noreen, 1998b), whereas the insoluble ethyl acetate extract was inactive. All the isolated compounds were tested for their activity in COX-1 and COX-2 assay and none of the compounds had PG specific activity.
The 1H NMR spectrum of crude ethyl acetate extract showed some interesting features in aromatic, olefinic and aliphatic methyl regions. From the most unpolar fractions compounds 1-4 (Fig. 1) were obtained. The molecular formula of 7-Hydroxy-1-oxy-14-norcalamenene 1 is C14H1802 with [M]+ at m/z = 218 and fragments at 176, 175, 159, 121, 91 and 77. The IR absorption spectrum suggested the presence of a,b unsaturated carbonyl group (1672 cm–1) and hydroxy group (3410 cm–1). The 1H and NMR spectra (Table 1 and 2) agreed with literature data (Bohlmann et al., 1979; Garcez et al., 1997). The mass spectrum of (+)-8-hydroxycalamenene 2 showed a molecular ion peak [M]+ at m/z = 218 corresponding to the molecular formula C15H220 and fragmentation patterns at 175, 160, 131 and 91. The IR spectrum showed the presence of hydroxyl group (3390 cm–1) and aromatic residue (1620 and 1460 cm–1). The 1H and 13C NMR spectra (Table 1 and 2) agreed with literature data (Nishizawa et al., 1983). The mass spectrum of T-cadinol 3 showed a molecular ion peak [M]+ at m/z = 222 corresponding to the molecular formula C15H26O2. The 1H and 13C NMR spectra (Table 1 and 2) agreed with literature data (Claeson et al., 1991). The mass spectrum of 7,14-dihydroxycalamenene 4 showed a molecular ion peak [M]+ at m/z = 234 corresponding to the molecular formula C15H22O2 and fragmentation patterns at 160, 131 and 91 and 77. The IR spectrum showed the presence of hydroxyl group (3360 cm–1) and aromatic residue (1618 and 1510 cm–1). The 1H and 13C NMR spectra (Table 1 and 2) agreed with literature data (Bohlmann et al., 1979; Garcez et al.,1997).
| Fig. 1: | Structures of the isolated compounds |
Discussion
According to the interesting features of 1H and 13C NMR spectrum of the EtOAc extract of S. macrotepala, MPLC have been carried out. From the earlier unpolar fractions four sesquiterpenes 1-4 (Fig. 1) have been obtained.
The nature of the sesquiterpene type of compounds 1, 2 and 4 was followed from 1H NMR spectra (Table 1). It showed that aromatic ring was substituted by hydroxy and methyl groups from d value at 6.39-7.44 ppm. Further more isopropyl function from methyne proton at d 1.89 -2.04 (m) and the methyl group as doublet at d 0.75-0.99 ppm where the methyl group attached to the aromatic ring at d 2.21-2.26 (s). The evidence of a cadinane-type or closely related carbon skeleton was deduced also from 13C NMR and DEPT spectra: Three methyl signals at d 15.68-22,18 ppm, two methylenes at d 19.18-35.28 ppm, three methynes at d 26.74 - 44.07 ppm and six aromatic carbons in which two were unsubstituted at d 112.33-123.09 ppm and one carried phenolic hydroxyl group at d 151.68-153.21 ppm, where the other carried methyl group at d 131.09-135.12 ppm.
The 1H NMR spectrum (Table 1) of compound 1 showed olefinic proton at d 5.56 (br s) and two methyl groups of isopropyl function located at d 0.78 and 0.90 (d). The 13C NMR spectrum (Table 2) showed the characteristic olefinic carbons at d 122.39 and 143.40 ppm and the peak at d 70.54 ppm indicated one hydroxyl group present in the molecule.
T-cadinol was shown to have a concentration-dependent smooth muscle relaxing effect on the isolated guinea pig ileum and a dose-dependent inhibitory effect on cholera toxin-induced intestinal hypersecretion in mice (Claeson et al., 1991) where ( +)-8-hydroxycalamenen-e showed not only significant toxicity against fish but also antibacterial activity (Nishizawa et al., 1983). The significant pharmacological activity of these compounds could explain the uses of S. macrotepala in traditional medicine.
Inspection of the above results reveals that studies on the secondary metabolites of the twigs and leaves of the Ecuadorian medicinal plant Siparauna macrotepala belongs to the family Monimiaceae led to isolation and characterization of 7-hydroxy-l-oxo-14-norcalamenene 1, (+)-8-hydroxycalamenene 2, (+)-T-cadinol 3 and 7,14-dihydroxycalamenene 4. The biological activity of compound 1 and 4 could explain the medical uses of Siparauna macrotepala in traditional medicine.
Acknowledgements
The author wish to thank Danida Project "Development of new Drugs against Hepatitis" under direction of Prof. I. Zeid for generous financial support. The author is very thankful to Prof. K. Torssell for permission to continue the work on the plant and to Dr. S. Khalifa for revision of the manuscript. The author is also very grateful to the Division of Pharmacognosy, Uppsala University, BMC, Sweden for performance of anti-inflammatory bioassays.