Modified Eremophilanes and Anti-Inflammatory Activity of Psacalium cirsiifolium

Quatro novos eremofilanos modificados, juntamente com dez derivados conhecidos de cacalol, dois cariofilenos, um aromadendreno e um flavonoide foram purificados a partir de Psacalium cirsiifolium. As estruturas destes compostos foram elucidadas por análise espectroscópica. A atividade anti-inflamatória dos extratos e de sete dos compostos isolados foi avaliada no modelo de 12-O-tetradecanoilforbol-13-acetato (TPA) de inflamação aguda induzida. O composto inédito 2a-hidroxiadenostin B (4) mostrou uma atividade dependente da dose (IC50 0,41 mmol por orelha) e um efeito de inibição de neutrófilos medido pelo teste de mieloperoxidase (MPO) semelhante ao efeito da indometacina, 0,31 e 1,0 mmol por orelha.


Introduction
Psacalium cirsiifolium is one of the 40 species of perennial herbs grouped into the genus Psacalium (Asteraceae, Senecioneae, Tussilagininae). 1 They are disseminated from the south of the United States to Guatemala and some of them are used in folk medicine to cure diabetes and renal, hepatic, gastrointestinal and dermatological problems. 2,3The hypoglycemic, anti-inflammatory and antioxidant activities of P. decompositum, [4][5][6] P. peltatum 7,8 and P. radulifolium 9 extracts have been reported.The antimicrobial effects of P. radulifolium 10 and the antiinflammatory properties of P. sinuatum 11 have also been determined.Sesquitepenes mainly of eremophilane and modified eremophilane types are the main secondary metabolites isolated from the eight species of the genus chemically studied so far: P. decompositum [4][5][6] (also studied as Cacalia decomposita), 12,13 P. tussilaginoides (studied as Cacalia ampulacea), 13 P. peltatum, 7,8 P. sinuatum, 11 P. radulifolium, 9,10 P. paucicapitatum, 14 P. megaphyllum 15 and P. beamanii. 16Cacalol has been identified as the major active compound in these species with antioxidant, 9 antimicrobial 10 and anti-inflammatory 17 activities.Cacalone, epi-cacalone, maturinone, and radulifolin D have also shown anti-inflammatory properties. 17,18As continuation of our research on Senecioneae we studied the chemical composition of P. cirsiifolium which to the best of our knowledge has no previous studies.We report the isolation of fourteen modified eremophilane derivatives (1-14), of which four (1-4) are described for the first time.Two known caryophyllenes (16,  18), one aromadendrene (17) and one flavonoid (15) were also isolated (Figure 1).The 12-O-tetradecanoylphorbol-13acetate (TPA) model of induced acute inflammation was used to evaluate the anti-inflammatory activity of extracts and of the isolated compounds non-evaluated previously.The most active compound (4) was tested on the myeloperoxidase (MPO) assay to determine its effect on the recruitment of inflammatory cells, such as neutrophils.

Results and Discussion
Compound 1 was obtained as a colorless oil.The IR spectrum indicated hydroxyl, ester and conjugated ketone groups (3390, 1743, 1661 cm -1 ).The molecular formula C 17 H 20 O 5 , determined by HRESIMS, showed eight degrees of insaturation.The 1 H NMR spectrum (Table 1) was similar to those of cacalone (5) and epi-cacalone (6) 6 with two additional signals at d H 5.13 (tt, 1H, J 6.0, 5.0 Hz) and 2.05 (s, 3H).The first one was attributed to H-2 by its correlations with the H 2 -1 and H 2 -3 methylene groups observed in the COSY experiment.The downfield chemical shift of H-2 indicated that an acetate group, whose methyl group appeared at d H 2.05 was attached to this position, and, in addition, H-2 showed an interaction with the carbonyl at d C 170.5 in the HMBC experiment.The NOESY spectrum showed interactions between H 3 -15 and the acetate methyl, therefore this group should have a b-pseudoaxial orientation, since on biogenetic grounds H 3 -15 is b. 19ikewise, the coupling constant of H-2 (J 6.0, 5.0 Hz) suggested its a-pseudoequatorial orientation.Moreover, a CD (circular dichroism) analysis of compound 1 showed a similar profile to that of epi-cacalone (6), consequently, the hydroxyl group at C-6 (d C 72.1) in 1 should be b-oriented as the one in 6.Therefore, 1 was identified as (2R, 4S, 6S)-2-acetoxy-epi-cacalone.
Compounds 2 and 3 exhibited the same molecular formula C 15 H 18 O 4 (HRESIMS) and very similar spectroscopic data, with evidence of hydroxyl (3400 cm -1 ) and conjugated carbonyl groups (1660 cm -1 ) in the IR spectra.The 1 H and 13 C NMR spectroscopic data of these compounds also resembled those of cacalone (5) and epi-cacalone (6), 6 and were indicative of the presence of an additional hydroxyl group.Position of this additional group at C-4 (d C 75.5 in 2 and 74.6 in 3) was supported by the correlations observed in the HMBC experiments between H 3 -15 and C-4 in both 2 and 3. Differences between the two compounds were however observed in their 1   was comparable with that of cacalone (5), indicating that they have the same stereochemistry at C-6 and, therefore, 2 and 3 are epimers at C-4.On the other hand, the fact that in compound 2, H 3 -14 and H 3 -15 resonated at lower field (Dd 0.06 and 0.11, respectively) than the same groups in 3, indicated that in compound 2, each of these methyl groups is feeling a deshielding effect due to its syn orientation with a hydroxyl group, which is not the case in compound 3. Additionally, in the carbon resonance of CH 3 -14 in 3 a downfield shift (Dd 3.7) was observed with respect to that in 2 as a result of a change from a pseudoaxial orientation in compound 2 to a pseudoequatorial in 3, in order to release steric crowding. 20In the case of CH 3 -15, this showed almost the same chemical shifts in 2 and 3 (Dd 0.01) since it adopted in both pseudoaxial orientation which was a in 2 and b in 3.
This last was evident by the NOESY interaction observed between H 3 -15 and H-1a in compound 2, and by the coupling in "M" between H 3 -15 and H-3a observed in the COSY experiment of 3. Therefore the absolute configuration of compounds 2 and 3 should be 4S, 6R and 4R, 6R, respectively.
The absolute stereochemistry of cacalone ( 5) and epi-cacalone( 6) has been determined 6 but, since their CD data were not available in literature, they were obtained in the present work.
The anti-inflammatory activity of the methanolic extracts of roots and aerial parts, and that of compounds 1-4, and 16-18 was evaluated using the TPA model of induced acute inflammation. 11Since the anti-inflammatory properties of compounds 5-15 had been previously reported, 11,17,18 they were not tested in the present work.As shown in Table 3, the extracts exhibited moderate activities (48.14 and 46.76% for roots and aerial parts, respectively).Compound 4, with 76.67% of edema inhibition, was the most active compound showing a dose dependent activity with IC 50 0.41 mmol per ear while that of the reference compound, indomethacin, was 0.24 mmol per ear (Table 4).
Myeloperoxidase (MPO) is a biochemical marker for tissue content of polymorphonuclear leukocytes because MPO activity is well correlated with the number of infiltrated cells in inflamed regions. 28,29In the MPO activity test compound 4 attenuated, in a dose-dependent manner, the activity of MPO and showed a similar effect to that of indomethacin at 0.31 and 1.0 mmol per ear (Figure 2).The Pearson's correlation analysis between skin weight and the MPO activity of all biopsies of 4 showed a positive correlation (r = 0.83, p < 0.001), indicating that edema inhibition of 4 is associated with the inhibition of infiltrated neutrophils in the ear biopsy.

General procedures
Melting points were determined on a Fisher-Johns melting point apparatus and are uncorrected.Optical rotations were determined on a Perkin-Elmer 343 polarimeter.Circular dichroism was obtained on a Jasco J-720 spectropolarimeter.UV and IR spectra were recorded on a Shimadzu UV 160U and a Bruker Tensor 27 spectrometer, respectively.1D and 2D NMR spectra were obtained on a Varian-Unity Inova 500 MHz spectrometer with tetramethylsilane (TMS) as internal standard.EIMS were determined on a Bruker Daltonics Analysis 3.2 mass spectrometer.HRESIMS were performed on a Bruker micrOTOF II mass spectrometer with mass resolution of 16.500 FWHM, mass interval 50-20,000 m/z, and speed 40 Hz.Column chromatography was carried out under vacuum (VCC) on silica gel G 60 (Merck, Darmstadt, Germany).Flash column chromatography (FCC) was performed on silica gel 230-400 (Macherey-Nagel, Germany).Sephadex column chromatography was developed with Sephadex LH 20 (Amersham Pharmacia Biotech AB, Sweden).Analytical TLC was carried out on silica gel 60 GF 254 or RP-18W/UV 254 (Macherey-Nagel, Germany) and preparative TLC on Si gel GF 254 layer thickness 2.0 mm or RP-18W/UV 254 layer thickness 1.0 mm.

Animals
Male NIH mice weighing 25-30 g were maintained in standard laboratory conditions in the animal house (temperature 27 ± 1 °C) in a 12/12 h light-dark cycle, being fed laboratory diet and water ad libitum, following the Mexican official norm MON-062-Z00-1999.

TPA-induced edema model
The TPA-induced ear edema assay in mice was performed as previously reported, 11 Tables 3 and 4.

Myeloperoxidase assay
Tissue MPO activity was measured in biopsies taken from ears 4 h after TPA administration using an adapted method of Bradley et al. 28 and Suzuki et al. 29 Each mouse ear biopsy was placed in 1 mL of 80 mmol L -1 phosphate-buffered saline (PBS) pH 5.4 containing 0.5% hexadecyltrimethylammonium bromide (HTAB).Each sample was homogenized for 30 s at 4 °C with a small sample laboratory Tissue Tearor Homogenizer (OMNI International, model 125).The homogenate was freezethawed at room temperature 3 times, sonicated 20 s and centrifuged at 12,000 rpm for 15 min at 4 °C.The resulting supernatants (10 mL in quadruplicate) were poured into 96 well microplate and 180 mL of 80 mmol L -1 PBS (pH 5.4) without HTAB were added.Microplate was heated at 37 °C then, 20 mL of 0.017% hydrogen peroxide were added to each well.For the MPO assay, 20 mL of 18.4 mmol L -1 3,3',5,5'-tetramethylbenzidine in 50% aqueous dimethylformamide were added to start the reaction.Microliter plates were incubated at 37 °C for 5 min.The reaction was stopped with 20 mL of 2 mol L -1 H 2 SO 4 .MPO enzyme activity was assessed colorimetrically using a BioTekMicroplate Reader (EL × 808) at an absorbance wavelength of 450 nm.MPO activity test results were expressed as percent of the maximal activity, Figure 2.

Statistical analysis
All data were represented as percentage mean ± standard error of mean (SEM).The statistical analysis was done by means of Student's t-test, whereas analysis of variance ANOVA followed by Dunnett test were used to compare several groups with a control.P values p ≤ 0.05 and p ≤ 0.01 were considered to be significant.Pearson's correlation coefficient was calculated for the edema and MPO results of compound 4.

Conclusions
This study shows that the modified eremophilanes are the main secondary metabolites in Psacalium cirsiifolium in agreement with the chemotaxonomy of the genus Psacalium reported so far.The study of the anti-inflammatory properties of seven of the isolated metabolites, using the TPA-induced mouse edema model, revealed that the new eremophilane derivative 2a-hydroxyadenostin B (4) was the most active compound and that this activity is associated with the inhibition of infiltrated neutrophils in the ear biopsy.

Figure 2 .
Figure 2. Comparative effect of 2a-hydroxyadesnostin B (4) and indomethacin on myeloperoxidase levels in the mouse ear edema induced by TPA.Data represent means ± standard error of five animals.*p ≤ 0.05 and **p ≤ 0.01.

Table 2 )
, the signals of C-14 and C-15 appeared at d C 28.5 and 29.6, respectively, while in compound 3 the signals of the same atoms were observed at d C 32.2 and 29.7.At this point, it was evident that 2 and 3 should have different stereochemistry.The CD spectroscopy of compounds 2 and 3

Table 4 .
Dose response evaluation of 2a-hydroxyadesnostin B (4) bEach value represents the mean of five animals ± standard error; a p ≤ 0.05; b p ≤ 0.01; c control 14.75 ± 1.13.