Bioactive new bitter-tasting p -hydroxystyrene glycoside and other constituents from the fern Elaphoglossum spathulatum

Two bitter-tasting compounds were isolated from an Argentine collection of Elaphoglossum spathulatum , a new p -hydroxystyrene glycoside that we named elaphoside-A, and naringin. Structure elucidation of the new compound was carried out on the basis of extensive two-dimensional 600 MHz NMR analyses, as well as high resolution-mass spectrometry and chemical degradation and derivatization. Moreover, we report herein the isolation of the hopanoid diploptene, stigmasterol, β -sitosterol, ergost-5-en-3-ol, phytol, aspidinol, desaspidinol, vitamin E, neophytadiene, 1-(2,4,6-trihydroxyphenyl)-2-pentanone, and 6,10,14-trimethyl-2-pentadecanone. In addition, we evaluated the alimentary and ovipositional response of Ceratitis capitata to the extracts and pure compounds of E. spathulatum . The new styrene glycoside produced severe disturbance in the ovipositional behavior of this pest insect.


Introduction
The chemistry of the around 3000 species of ferns (pteridophytes) growing in Central and South America has been scarcely investigated.The genus Elaphoglossum Schott 1 is widely distributed in these regions and some of its species are endemic and cover humid rocky walls in the Northwest of Argentina.

Results and Discussion
The air-dried sterile fronds were ground and extracted with diethyl ether and then methanol.The diethyl ether extract was analyzed by GC─MS to detect the common plant constituents βsitosterol, stigmasterol, stigmast-5-en-3-ol, ergost-5-en-3-ol, stigmast-4-en-3-one, phytol, neophytadiene, vitamin E, methyl arachidonate, 6,10,14-trimethyl-2-pentadecanone and 1-(2,4,6trihydroxyphenyl)-2-pentanone.Three compounds, characteristic of ferns, were also detected, aspidinol (1), desaspidinol (2) and the hopanoid diploptene (3).Crystalline diploptene was further isolated from the ether extract employing chromatographic methods, and identified by its melting point, [α] D , and spectroscopic features, in comparison with available data in the literature. 7It is important to point out that hopanoids are a group of pentacyclic triterpenoids only found in ferns, bryophytes, and bacteria 6 .Aspidinol and desaspidinol seem to be responsible for the anthelmintic effects detected in extracts from various species of ferns of the genus Dryopterys. 2 The crude methanol extract was repeatedly chromatographed on silica gel and Sephadex LH-20, to yield two bitter glycosides, the new elaphoside-A (4), and the known naringin (5) a flavanone neohesperidoside frequently found in citrus.
The FT-IR spectrum of elaphoside-A (4) showed a broad absorption at 3338 cm -1 due to the O-H stretching, a C=C band at 1628 cm -1 as well as two intense C-O bands at 1080 and 1044 cm -1 .The positive HR-FAB-MS spectrum of 4 gave a quasimolecular ion peak [M+Na] + at m/z: 467.1538, consistent with the molecular formula C 20 H 28 O 11 , which accounts for seven degrees of unsaturation.The UV absorption pattern indicated the presence of a p-O-substituted styrene residue as has been reported for ptelatosides (styrene glycosides) isolated from the fern Pteridium aquilinum var.latiusculum. 2The 600 MHz 1 H-NMR spectrum displayed two doublets at δ 7.35 and 7.04 (J = 8.4 Hz) consistent with the presence of four aromatic protons on a pdisubstituted benzene ring, as well as evidence for a monosubstituted double bond attached to a benzene ring: three signals at δ 6.66 (dd, J cis = 10.8Hz, J trans = 17.4 Hz), 5.64 (dd, J trans = 17. 4  Hz, J gem = 0.9 Hz) and 5.11 (dd, J cis = 10.8Hz, J gem = 0.9 Hz).The HMQC ( 1 H─ 13 C correlation) provided evidence for seven oxygenated methines and two oxygenated methylenes.The presence of a sugar moiety was clear by the signals between δ 3.3 and 5.2 ppm in the 1 H spectrum as well as the 13 C signals between δ 68 and 106 ppm, two anomeric C being detected.Correlations were found between C-1 of the styrene moiety (δ 158.8) and the anomeric proton of allose (δ 5.19, d, J = 7.8 Hz) and between C-3 of allose (δ 83.9) and the anomeric proton of glucose (δ 4.53, d, J = 7.8 Hz) in the heteronuclear multiple bond correlation spectrum (HMBC) of 4. NOESY cross peaks were observed indicating a space proximity of aromatic protons (δ 7.4, d, J = 8.4 Hz) and the anomeric proton of allose as well as the H-3 of allose (δ 4.31, t, J = 3 Hz) and the anomeric H of glucose.The mentioned evidence revealed the location of the sugar and the nature of the disaccaride.Acetylation of 4 gave the heptaacetylated derivative (6).Configurations of the sugar moieties at the anomeric protons were determined to be βbased on the coupling constants of the anomeric protons (J 1',2' = J 1'',2'' = 7.8 Hz) in 4 and 6.Acidic hydrolysis of 4 afforded glucose, allose and p-hydroxystyrene.HPLC analysis of sugars employing a chiral detector provided their absolute configurations.Further evidence was accomplished by methanolysis followed by acetylation of 4 that gave a mixture of methyl-2,3,4,6-tetra-O-acetyl-β-D-allopyranoside, methyl-2,3,4,6-tetra-O-acetyl-α-D-glucopyranoside, and the methanol adduct of the aglycone phydroxystyrene (7) 8 .Thus the structure of 4 is represented as p-hydroxystyrene Identification of naringin ( 5) was accomplished by means of chemical evidence and comparison of its spectral features with literature data 9 .Acid hydrolysis of naringin followed by HPLC analysis employing a chiral detector afforded the aglycone 4',5,7-trihydroxyflavanone, Lrhamnose and D-glucose.The sugar moiety was determined to be neohesperidose (2-O-α-Lrhamnopyranosyl-D-glucopyranose) by HMBC and NOESY spectra.Acetylation of 5 gave the hexaacetylated derivative 8.
So far, reports on the natural occurrence of p-hydroxystyrene and its glycosides is rare.While p-hydroxystyrene and its β-D-glucoside were isolated from Papaver somniferum in 1945, 10 the isolation of a few p-hydroxystyrene glycosides from ferns was described many years later, in 1985. 2 Noteworthy, the disaccharide neohesperidose has been found in other ferns as the sugar moiety of p-hydroxystyrene glycosides while in our present collection it is attached to the flavanone naringenin.

Effects of E. spathulatum constituents on the ovipositional behavior of C. capitata
The fruit fly C. capitata causes important economical damages in the north of Argentina.Substances that affect their behavior can be used in the development of pest control agents.Crude extracts and bitter substances of E. spathulatum were tested to evaluate their effects on the ovipositional behavior of C. capitata.
The influence of chemical agents on the ovipositional response of the fruit fly Anastrepha suspensa (Loew) was studied by Szentesi et al. in 1979. 11 As a part of their study, they evaluated the effect of naringin, and found that females did not respond differentially to plain or treated agar, except for the highest concentration tested (0.01 M) in which females rejected treated agar.
Our quantitative results are described herein employing an oviposition index (I O = 100T/C) calculated as the ratio of the number of eggs laid inside the treated substrate (T) and the ones laid inside the control substrate (C) expressed in %.The results of this experiment are summarized in Table 1.Pair wise Tukey test indicated a significant difference in the preference for the control substrate compared with the substrate treated with 6 µg/cm 2 of elaphoside-A and 14 µg/cm 2 of ether or methanol extract.Naringin did not significantly affect the fruit fly behavior at the tested concentration (P>0.05 are considered not significant).

Diet choice test
When treated and control diets were presented to C. capitata adults, no significant differences were observed in their response.Treated diets contained 250 ppm of extracts and bitter pure compounds obtained from E. spathulatum.

Experimental Section
General Procedures.For TLC detection, Godin reagent 12 was used.For separation of mixtures an HPLC equipped with differential refractometer, a silica gel and a reverse phase C18 columns were employed.For separation of sugars a Shodex Rspak NH2P-50 4E column.Absolute configuration of sugars was determined using chiral detection in a Shodex OR-1 detector.The mixture CH 2 Cl 2 -MeOH (1:1) was used for separations with Sephadex LH-20.GC-MS spectra were obtained on a Hewlett Packard 6890 gas chromatograph with an HP-5973 mass selective detector.A capillary column (30m x 0.25µm i.d.) coated with phenyl methyl siloxane (0.25µm film thickness) was used.IR spectra were recorded on a JASCO FT/IR-41.Ultraviolet spectra were measured in methanol on a SHIMADZU UV-160A spectrophotometer.NMR spectra were performed at 150 and 75 MHz for 13 C and 600 and 300 MHz for 1 H on a Varian UNITY 600 and Varian MERCURY 300, respectively.Optical rotations were measured on a JASCO DIP-1000 automatic digital polarimeter.
Plant material, extraction and isolation.Elaphoglossum spathulatum was collected in June of 2001 at Tiraxi, Jujuy province, Argentina.A voucher specimen (Muruaga 366) was deposited in the Herbarium of the Fundación Miguel Lillo, Tucumán, Argentina.
After being air-dried, the sterile fronds (30 g) were mechanically powdered and then successively extracted with ethyl ether and methanol.
A portion (3 g) of the methanol extract (5.5 g) was repeatedly chromatographed on silica gel with CHCl 3 -MeOH mixtures, and Sephadex LH-20, leading to the isolation of elaphoside-A (4, 37 mg) and naringin (5, 124   Methanolysis of elaphoside-A (4): A solution of 4 (17.8 mg) and concentrated H 2 SO 4 (20 µl) in MeOH (4 ml) was refluxed for 10 hours, cooled to room temperature, neutralized with a saturated NaHCO 3 solution, and concentrated.The residue was diluted with water (3 ml) and the mixture was extracted with CHCl 3 (3x3 ml).The combined organic extracts were dried and concentrated to give a phenol (7, 2 mg).The spectral data of this compound was in good agreement with data from literature 8 .The aqueous layer of the reaction mixture was concentrated.A solution of the residue and Ac 2 O (2 ml) in pyridine (2 ml) was stirred at room temperature for 14 hours and concentrated.The residue was extracted with CHCl 3 (5 ml)

Insects.
A colony of C. capitata was initiated with pupae obtained from infested oranges from the Northwest of Argentina.Adults fed on a diet made of water and a mixture of sugar and yeast hydrolysate (3:1).They were maintained in a rearing room with a photoperiod 12L:12D, at 24 ± 2ºC and 60 ± 10 % RH. 15 Bioassays.Ovipositional behavior: Oviposition substrates were prepared with a mixture of peach juice (500 ml), agar (1 teaspoonful) and sodium benzoate (1 teaspoonful) as preservative.This agar solution was poured into cylindrical molds, allowed to gel, and finally sliced.Agar cylinders were then wrapped in film paper to avoid dehydration.The surface of the wrapped cylinders was pricked with a needle and treated with an acetone solution of the sample to be tested in order to deposit 6 µg/cm 2 of pure compounds or 14 µg/cm 2 of crude extracts.Control cylinders were treated with acetone and the solvent was then eliminated in vacuo.Three groups of C. capitata adults were selected from our laboratory colony.Each group, consisting of five male-female pairs, was placed in a small cage, covered with a voile.Two agar cylinders (sample and control) were placed on the voile.After 4 days, eggs were gently rinsed from the agar and counted.The experiment was conducted in 3 replicates.
Diet choice test: Treated diets were prepared by impregnating a 3:1 mixture of sugar and yeast hydrolysate with an acetone solution of the sample to be tested to obtain a final concentration of 250 ppm.Control diet was only impregnated with the solvent, which was then evaporated in vacuo.Two dishes, containing treated and control diets, were placed in each cage.20 adult couples were released in each cage.After 10 min the landings of individuals on each diet were counted during a 2 hours period.This bioassay was made by triplicate.
Statistical Analysis.Results are reported as mean ± SEM.Differences in the mean values were evaluated by analysis of variance (ANOVA).The Tukey test was used for all pair wise comparisons of groups.In all statistical analysis P values > 0.05 were considered not significant.

Table 1 .
Oviposition index (I O ) of crude extracts and bitter-tasting compounds isolated Means within a column or a row followed by the same letter (lower and upper case respectively) are not significantly different (P>0.05,Tukey multiple range test).
a b Numbers represent mean ± SEM; n = 3.