Sesquiterpene Lactones and other Chemical Constituents of Mikania hoehnei R .

O estudo fitoquímico de Mikania hoehnei conduziu ao isolamento e à identificação de acetato de lupeol, estigmasterol, β-sitosterol, campesterol, 3-O-β-D-glicopiranosil estigmasterol, 3-O-β-Dglicopiranosil sitosterol, 2,6-dimetoxibenzoato de benzila, luteolina, caempferol e duas lactonas sesquiterpênicas diidrocostuslactona e 8β-hidroxizaluzanina D. Estas substâncias foram identificadas com base na análise dos espectros de IV, EM e RMN de H e C e os dados foram comparados com os descritos na literatura.


Introduction
Mikania hoehnei B. Robinson, first described by B. Robinson in 1934, is an endemic vine found in Brazil from Rio de Janeiro to Santa Catarina. 1,2Only one report has appeared dealing with the terpenoids of M. hoehnei, 3 in which the presence of lupeol and stigmasterol was indicated.][6] The genus Mikania has undergone taxonomic studies, where morphologic information was considered 1,[7][8][9] and an evaluation of the existence of correlations between the terpenoid chemistry and the phylogeny of those species was done. 10,11Mikania hoehnei B. Robinson belongs to the subtribe Mikaniinae, section Mikania (L.) Willd.(Holmes, private communication) and phytochemical investigation of the whole plant led to the isolation of two sesquiterpene lactones.][14][15][16][17] Although the occurrence of sesquiterpene lactones is common in species of Mikania, guaianolides have been found in only 3 of the 46 species studied so far, e.g. in M. vitifolia, 18 M. haenkeana 19 and M. mendocina. 20There also seems to be a remarkable difference between the compounds identified in M. hoehnei and those reported for other Mikania species.

General
The IR spectra were obtained on NaCl film in a Perkin Elmer model 1420 spectrophotometer. 1 H NMR (300 MHz) and 13 C NMR (75 MHz) spectra were recorded on a Bruker DPX 300 in CDCl 3 with TMS as internal standard.EIMS was obtained at 70 eV on HP 5988-A.Prep.TLC was carried out on Si gel PF-254 (Merck), CC on Si gel 60 (0.063 a 0.200) (Merck) and VLC on Si gel 60 H (0.005 -0.045) (Merck).

Plant material
Mikania hoehnei B. Robinson was collected in Restinga de Maricá, Rio de Janeiro, RJ, Brazil, in July 1996, and identified by Professor Dr. Janie G. Silva (Instituto de Biologia da Universidade Federal Fluminense, Rio de Janeiro).A voucher specimen (SPFR 04309) was deposited in the herbarium of the Department of Biology, FFCLRP/ USP and was used for the authentication of the species.

Extraction and fractionation
Dried and powdered whole M. hoehnei plants (2.8 kg) were exhaustively extracted at room temperature with hexane, ethyl acetate and ethanol in successive phases.Evaporation of solvents under reduced pressure furnished 64.0 g, 25.0 g and 35.0 g respectively, of crude extracts.
The bulk of the hexane extract (60.0 g) was chromatographed over silica gel under vacuum (VLC) and eluted with hexane, gradually increasing the polarity with ethyl acetate and then methanol.Twelve fractions were collected.Fraction 2 (60.0 mg) was submitted to prep.TLC (silica gel), eluting with hexane-ethyl acetate 9:1 (v/v), to afford 16.0 mg of lupeyl acetate.CC of fraction 10 (600.0 mg) on silica gel (hexane, ethyl acetate and methanol in mixtures of increasing the polarity) followed by precipitation (methanol) of subfr.10.13 (45.0 mg) yielded 5.0 mg of a mixture of β-sitosterol, campesterol and stigmasterol and 5.3 mg of a mixture of β-sitosteryl glucopyranoside and stigmasteryl glucopyranoside.
The crude ethyl acetate and ethanol extracts were separately suspended in MeOH-H 2 O 19:1 (v/v).The solutions were partitioned first with hexane and then with dichloromethane.After evaporation at reduced pressure, the dichloromethane fractions were chromatographed separately on Si gel 60 (CC) with hexane and gradually increasing polarity, with ethyl acetate and then methanol.All fractions monitored by TLC.
The hydroalcoholic extract (2.0 g) from the crude ethanol extract, soluble in methanol, was chromatographed on a Sephadex LH -20 column using methanol as eluent, and twelve fractions were collected.Fraction 4 yielded 15.0 mg of luteolin and fraction 7 yielded 4.0 mg of kaempferol.

Results and Discussion
The chromatographic fractionation of hexane, ethyl acetate and ethanol extracts yielded stigmasterol, β-sitosterol and campesterol, lupeyl acetate, 21 β-sitosteryl and stigmasteryl glucopyranosides, 22 benzil 2,6-dimethoxybenzoate, 23 luteolin, 24 kaempferol 24 and dehydrocostuslactone. 13,14 The structures were established by comparison of their spectroscopic properties (mainly IR, 1 H and 13 C NMR) with those reported in the literature and in some cases by direct comparison with authentic samples.
Structure of the guaianolide 1 was deduced from the IR, MS, 13 C and 1 H NMR spectra data and the stereochemistry was defined from the coupling constants' value J and nOe difference correlations.This lactone has been previously reported as a synthetic compound obtained in the controlled acetylation of the natural guaianolide Integrifolin (8-epi-desacylcynaropicrin). 12The IR spectrum displayed bands at 1750 cm -1 (γ-lactone), at 1730 cm -1 (acetyl group), at 3494 cm -1 (hydroxyl group) and weak bands at 1664, 1641 cm -1 (c=c double bonds).
Inspection of the 1 H NMR spectrum of compound 1 indicated several signals very close to those observed in Zaluzanin D 15 .The position and the stereochemistry (βorientation) of hydroxyl group at C-8 were determined by nOe correlation (Figure 1).nOe's were observed between the H-8 (δ 4.37) and H-13a (δ 5.62), H-7 (δ 2.98) and H-9b (δ 2.61).In the same way, correlation of the H-3 (δ 5.50) with H-2b (δ 2.45) and H-15a (δ 5.32) in the nOe spectrum confirmed the α-orientation of H-3 which was attached to the carbon atom which is linked to the acetate group, as previously defined as Zaluzanin D (compound 2).
Just as the NMR spectrum of 2, the NMR spectrum of guaianolide 1 exhibited two characteristic doublets at δ 6.42 and 5.62 (J 3.8 and 3.2 Hz respectively) corresponding to hydrogens of H -13a and H -13b of an exocyclic methylene group conjugated with a γ-lactone.The exocyclic methylene groups attached to C-10 and C-4 were characterized by two singlets at δ 4.99 (H-14a), 5.10 (H-14b) and a pair of triplets (J 2.0 Hz) at δ 5.32 (H-15a) and 5.52 (H-15b), respectively.
The stereochemistry cis of the ring junction at C-1 and C-5 was established by the coupling constant (J 8.3 Hz) between H-1 and H-5.In the case of 1β, 5α-transguaianolide, a value of 10.0 Hz or greater is expected according to that reported in the literature. 25,26The lactone ring junction was confirmed to be trans (6β H, 7α H) by the large coupling constant (J ~ 3.0 Hz) of the H -13-αmethylene protons this is due to H-6/H-7 (J 8.9 Hz) coupling constant. 16ll other signals were in agreement with the proposed structure of the guaianolide 1 (Table 1).Heteronuclear multiple quantum correlation 1 H -13 C (HMQC) allowed us to assign unambiguously the signals of all carbons.
Besides the importance chemosystematic, 27 sesquiterpene lactone posseses a wide spectrum of biological activity. 28Zaluzanin C, Zaluzanin D and its derivatives have shown several biological activities, such as activity against the P 388 lymphocytic leukemia in vitro, 29,30 antifungal activity, 31,32 inhibitory activity on nitric oxide production and nuclear fator KB, 33 inhibitory activity on ethanol absorption. 34n the basis of the above data, we conclude that M. hoehnei belongs to the Brazilian Mikania species groups, which produce sesquiterpene lactones.As other authors have also reported, 4,11 we conclude after analysis of all this information that it is too early to establish correlations between the terpene chemistry and the morphology of this huge genus.

Table 1 .
1H NMR data (δ) for 1 at 300 MHz a Signals attributed according spectrum in C 5 D 5 N. Figure 1.nOe correlations of compound 1.