Chemical Constituents , Antiproliferative and Antioxidant Activities of Vernonanthura nudiflora ( Less . ) H . Rob . Aerial Parts

Sesquiterpene lactones are an important class of secondary metabolites frequently isolated from Vernonanthura genus that present a variety of biological properties, including antiproliferative activity. Due to the limitation of pharmacological studies on Vernonanthura nudiflora, the aim of this work was to investigate their antioxidant potential and antiproliferative activity against human tumor cells, as well as to isolate and identify the chemical constituents present in their aerial parts. The phytochemical investigation resulted in the isolation of the sesquiterpene lactones piptocarphins A, B, D, and a new hirsutinolide derivative, 8α-tigloyloxy-10α-hydroxy-hirsutinolide, besides triterpenes, glycosylated steroids, flavonoids, and one chlorogenic acid derivative. Also, other sesquiterpene lactones were identified by ultra-high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) from dichloromethane fraction. This fraction showed activity against the tumor cells tested, mainly against leukemia, glioma, ovarian and kidney, with growth inhibitory activity (GI50) less than 0.80 μg mL. Piptocarphins A and B, in mixture, showed strong activity against all human cancer cell lines tested, with GI50 values ≤ 0.15 μg mL. Piptocarphin D was selective for glioma and resistant ovarian cell lines. The new hirsutinolide derivative showed potent activity against breast (GI50 = 0.96 μg mL) and resistant ovarian (GI50 = 3.60 μg mL) cell lines.


Introduction
Vernonanthura H. Rob.(Asteraceae family) comprises approximately 70 species widely distributed from southern Mexico to central Argentina, [1][2][3][4] of which 41 occur in Brazil, concentrated mainly in South and Southeast regions. 5ome species now assigned to Vernonanthura were originally placed in Vernonia genus.Robinson 4 segregated most of the South America species in 22 new genera, including Vernonanthura.Several Vernonanthura species are known in Brazil as "assa-peixe", and are used in traditional medicine for the treatment of flu and colds. 6,7pecies of this genus have also been reported to possess pharmacological activities, 8 such as antiplasmodial, 9 antileishmanial, [10][11][12] antimicrobial, [13][14][15] antioxidant, 11 antinociceptive and anti-inflammatory. 16,17ernonanthura nudiflora (Less.)H. Rob., popularly known as "alecrim do campo", is a sub-shrub, 50-80 cm high, flowering showy, native to South America, with distribution in Argentina, Brazil and Uruguay. 5,18,19A provoked experimental intoxication in sheep showed that V. nudiflora has moderate toxic action on the digestive system of this animal, nevertheless no significant effects on the respiratory and circulatory system were observed. 20revious experiment undertaken by Dobereiner and Tokarnia 21 also showed that V. nudiflora provoke irritation on the digestive tract mucosa of cattle and sheep.
V. nudiflora ethanolic extract presented potent antiproliferative activity against leukemia tumor cell lines, evidencing promising therapeutic potential. 22Previous phytochemical investigation of this species revealed the presence of triterpenes, steroids, flavonoids, and mainly sesquiterpene lactones of glaucolide, hirsutinolide and cadinanolide classes. 23,24Sesquiterpene lactones are still an important class of secondary metabolites, providing new therapeutic leads, especially for development of anti-inflammatory and anticancer agents. 8,25,26irsutinolide-type sesquiterpene lactones isolated from natural products, and semi-synthetic analogues, showed in vitro and in vivo activity against human glioma cell lines. 27dditionally, Youn et al. 28 reported that hirsutinolides isolated from Vernonia cinerea were able to inhibit glioblastoma (U251MG), and breast tumor cells (MDA-MB-231), which demonstrate the potential of this class of compounds.
In this paper, we describe the isolation and structural elucidation of one new hirsutinolide derivative, along with sixteen known compounds.In addition, other nine sesquiterpene lactones were identified by ultra-high pressure liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS/MS) from dichloromethane fraction.Moreover, due to the limitation of pharmacological studies on V. nudiflora, the evaluation of their antioxidant potential, and antiproliferative activity against human tumor cells, were carried out.

General experimental procedures
Chromatography separations were performed on silica gel 60 (70-230 mesh, Merck), silica gel flash (230-400 mesh, Acros Organics), Sephadex LH-20 ® (Sigma) or polyvinylpolypyrrolidone (PVPP, Sigma-Aldrich) chromatography columns (CC).Thin layer chromatography (TLC) was performed on normal phase pre-coated silica gel 60G or 60GF 254 (Merck) plates.Visualization of the compounds on TLC was accomplished by UV irradiation at 254 and 366 nm, and/or by spraying with H 2 SO 4 / anisaldehyde/acetic acid (1: 0.5: 50 mL) solution followed by heating at 100 °C.HPLC separations were performed on a Shimadzu instrument (Mod.Prominence) with two LC-20AR pumps, degasser DGU-20ASR, detector UV-Vis SPD-M20A model and injection system automatic SIL-10AF, and equipped with a Shim-pack PREP-ODS (250 × 20 mm; 15 μm) column.The mobile phase consisted of water (Milli-Q, Millipore) and methanol (Sigma Chemicals Co).Nuclear magnetic resonance (NMR) spectra were recorded on a VARIAN Mercury Plus spectrometer operating at 300 and 75.5 MHz, and Bruker avance III HD spectrometer operating at 500 and 125 MHz, using CDCl 3 and dimethyl sulfoxide (DMSO-d 6 ) as solvents.The UHPLC analysis was performed in a Shimadzu Nexera X2 instrument, equipped with a CBM-20A a system controller, two LC-30AD pumps, a CTO-30A column oven and SIL-30AC autosampler.The mass spectra were recorded on Bruker IMPACT II mass spectrometer, with electrospray ionization source (ESI) in the positive ion mode, quadrupole-time of flight (Q-TOF) analyzer and multichannel plate (MCP) detector.The optical rotation was measured on a PerkinElmer polarimeter at 24 °C and λ = 589 nm (sodium d-line), using a 1 cm microcell (c 0.15, CHCl 3 ).UV-Vis spectra were recorded using a PHARO 300 spectrophotometer (Merck).The solvent was removed using Rocket Synergy sample concentrator (Genevac).

Analysis of the dichloromethane fraction by UHPLC-HRMS/ MS
The sample was prepared in MeOH (1.0 mg mL -1 ) and chromatographic separations were performed using UHPLC on a Symmetry C18 column (75 × 2.0 mm i.d.; 1.6 μm Shim-pack XR-ODS III), maintained at a temperature of 40 ºC.The mobile phase consisted of H 2 O (solvent A) and 0.1% formic acid in CH 3 CN (solvent B).The gradient program was as follows: initial 0-1 min, using elution A-B (95:5, v/v), 1-3 min (30:70 v/v), 3-12 min (5:95 v/v) and kept at 95% B for 16 min at a flow rate of 0.2 mL min -1 .Injection volume was 3 μL.High resolution mass spectrometry analysis were carried out in a Q-TOF mass spectrometer via an electrospray ionization interface.The capillary voltage was operated in positive ionization mode, set at 4500 V, using sodium formate (10 μM) as calibrant.The dry gas parameters were set to 8 L min -1 at 200 ºC with a nebulization gas presure of 4 bar.Colision-induced dissociation (CID) fragmentation was performed using argon (Ar) collision gas and collision energy from 0-30 eV.Spectra data of the investigated compounds were collected from m/z 50-1300 with a resolution of 50.000, and with an acquisition rate of 5 spectrums per second.The ions of interest were selected by auto MS/MS scan fragmentation.The data processing software was Data analysis 4.3 (Bruker).Moreover, the mass error value was calculated.Only molecular formulas ≤ 5 ppm of error were considered in this study. 29

In vitro antiproliferative assay
In vitro antiproliferative activity experiments on human cell lines were performed according to Monks et al. 30 The crude extract and fractions of V. nudiflora were evaluated in vitro against ten human tumor cell lines [U251 (glioma, CNS), UACC-22 (melanoma), MCF-7 (breast), NCI-ADR/RES (ovarian expressing the multiple drug resistance phenotype), 786-0 (renal), NCI-H460 (lung, non-small cells), PC-3 (prostate), OVCAR-3 (ovarian), HT-29 (colon), and K-562 (leukemia)], kindly provided by the National Cancer Institute (Frederick, MA, USA).More, some of the isolated compounds (6 + 7, 8, 9, 10) were tested against six human tumor cell lines [U251 (glioma, CNS), MCF-7 (breast), NCI-ADR/RES (ovarian expressing the multiple drug resistance phenotype), NCI-H460 (lung, non-small cells), PC-3 (prostate), HT-29 (colon)].All samples were also evaluated against the immortalized human keratinocytes (HaCat) cell line (provided by Prof Dr Ricardo Della Coletta, UNICAMP).Stock solution (0.1g mL -1 ) of each samples was prepared in DMSO and successively diluted in RPMI 1640, supplemented with 5% fetal bovine serum and 1% penicillin:streptomycin mixture (1000 UI mL -1 :1000 μg mL -1 ) to final concentrations (0.25, 2.5, 25 and 250 μg mL -1 for the extract and fractions, and 0.15, 1.50, 15.0 and 150.0 μg mL -1 for the isolated compounds).The chemotherapeutic doxorubicin chloridrate (0.025, 0.25, 2.5 and 25 μg mL -1 ) was used as a positive reference standard to determine the sensitivity of cell lines.Cells in 96-well plates (100 μL cells per well) were exposed to sample concentrations, in triplicate, for 48 h at 37 °C and 5% of CO 2 .The final DMSO concentration (≤ 0.25%) did not affect cell viability.Before (plate control) and after sample addition, cells were fixed with 50% trichloroacetic acid, and cell proliferation was determined by spectrophotometric quantification of cellular protein content at 540 nm (Molecular Devices, model VersaMax) using sulforhodamine B. Two effective concentrations, named growth inhibition 50% (GI 50 ) and total growth inhibition (TGI), were calculated by non-linear regression analysis (sigmoidal fit) using Origin 7.5 ® (OriginLab Corporation). 31The selectivity index (SI) was calculated as SI = GI 50 HaCat / GI 50 tumor cell line. 32 vitro antioxidant assays DPPH free-radical scavenging activity The free radical scavenging effect of the crude extract and fractions of V. nudiflora was investigated using the DPPH (1,1-diphenyl-2-picrylhydrazyl) (Sigma-Aldrich) assay, based on the method proposed by Boroski et al. 33 with slight modifications.Serial concentrations (5, 10, 25, 50, 75, 100, 150 and 250 μg mL -1 ) of extract and fractions were prepared by addition of 20 mg of samples and 10 mL of methanol and 2 mL of DPPH methanolic solution (3.20 mg DPPH in 100 mL) were mixed with these solutions.The mixture was thoroughly vortex-mixed and kept in the dark for 30 min.Absorbance was measured using a UV-Vis spectrophotometer at 517 nm.Rutin and ascorbic acid were used as standards antioxidant compounds.All samples were tested in triplicate.The results were expressed as percent inhibition of the DPPH radical, which was calculated using the equation 1: where A DPPH is the absorbance of the DPPH solution and A sample is the absorbance of the DPPH solution with the sample tested.The sample concentration that afforded 50% inhibition (IC 50 ) was obtained by plotting the concentrations of the sample solutions versus percent inhibition.

ABTS •+ radical scavenging activity
The ABTS method was performed according to method described by Re et al. 34 with some modifications.Briefly, 7.0 mM of ABTS (2,2-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid) (Sigma-Aldrich) and potassium persulfate (140 mM) were mixed, and keeping in the dark for 16 h at room temperature.For the analysis, the ABTS •+ solution was diluted in ethanol (PA) to afford the ABTS •+ work solution (absorbance of 0.700 ± 0.050 at 734 nm).Aliquots (30 μL) of each extract and fractions, at different concentrations in triplicate, were homogenized with ABTS •+ work solution (3 mL) and, after 6 min, the absorbance was measured at 734 nm using a UV-Vis spectrophotometer.The results were expressed as percent inhibition of the ABTS radical, using the equation described in the DPPH assay.Trolox (6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid; Sigma Aldrich) was used as antioxidant standard.

Total phenolic compounds (TPC)
The total phenolic content (TPC) was determined using the Folin-Ciocalteu method as described by Singleton and Rossi, 35 and adapted by Boroski et al. 36 One aliquot (0.25 mL) of each sample in MeOH (2.5 mg mL -1 ) was diluted in distilled water (4 mL), and mixed with Folin-Ciocalteu reagent (0.25 mL) and saturated sodium carbonate solution (0.50 mL).This mixture was kept in the dark at room temperature (23-25 ºC) for 1 h before the absorption measurement at 765 nm.Gallic acid was used as standard in the calibration curve.Results were expressed as milligrams of gallic acid equivalents per gram of sample (mg GAE g -1 extract).

Total flavonoids (TF)
The total flavonoid (TF) content of crude extract and fractions was determined by aluminium chloride colorimetric method, 37 adapted by Boroski et al. 36 Aliquot (0.5 mL, in triplicate) of each sample diluted in MeOH (2.5 mg mL -1 ) was mixed with 5% AlCl 3 solution (0.25 mL) and methanol (4.25 mL) in graduated centrifuge tubes (15 mL).The resulting mixture was allowed to rest for 30 min at room temperature, protected from light.After that, the absorbance at 415 nm was measured.Quercetin was used as standard.
The flavonoid content was expressed in mg of quercetin equivalent per gram extract (mg QE g -1 extract).

Statistical analysis
The results were submitted to variance analysis (ANOVA) and Tukey's test (5% probability) using the software Assistat ® (version 7.7). 38
Structural elucidation of 8α-tigloyloxy-10α-hydroxyhirsutinolide (9)   Compound 9 was obtained as a colorless gum, [α] D 24 -5), all these signals corroborate the skeleton of a hirsutinolide-type sesquiterpene. 40,42,51In addition, the 1 H and 13 C NMR spectra showed an olefinic signal at d H 7.06 (q, J 7. 3) permitted assignment of the tigloyl group in the structure (Figure 2).The attachment of this group at C-8 in compound 9 was supported by the NMR chemical shifts values of H-8 reported for hirsutinolides esterified in the C-8 position (d H 6.36 to 6.60) 40,42 and non-esterified (d H 5.46). 40,54The signal for H-8 of compound 9 appears in d H 6.60, which is consistent with the linkage of tigloyloxy group at C-8.
NOESY spectra showed correlations between the hydroxyl groups at d H 4.11 (OH-1) and d H 3.88 (OH-10) with the methyl group at d H 1.57 (CH 3 -15).Besides that, correlations of H-8 (d H 6.60) with H-9β (d H 2.61), and between H-9β and methyl group CH 3 -14 (d H 1.25), indicated that H-8 is in the β position (Figure 2).Moreover, previous studies have reported that 8S* configuration appears to be generally the most accepted for hirsutinolidestype with H-8 in the β position. 41,42,51dentification of sesquiter pene lactones in the dichloromethane fraction by UHPLC-HRMS/MS The dichloromethane fraction (DC) of V. nudiflora was also analyzed by UHPLC-HRMS/MS (positive ion mode), using the sesquiterpene lactones 6-9, previously isolated and characterized by NMR analysis as standards.This strategy allowed the identification of more nine sesquiterpene lactones (Figure 3, Table 2).

Antiproliferative activity
The antiproliferative activity of the crude extract, fractions and isolated compounds of V. nudiflora aerial parts were described as GI 50 , TGI and SI values (Tables 3  and 4).To analyze these results, we assumed the criteria described by Fouche et al. 58 that stablished the values of GI 50 ≤ 30 μg mL -1 and TGI ≤ 50 μg mL -1 for promisor antiproliferative samples.
4][65] Therefore, the high antiproliferative potential observed for DC fraction can be partially justified by the presence of hirsutinolide sesquiterpene lactones, toghether with flavonoids and steroids that in association may have contributed to final effect.
Considering of the ABTS and DPPH assays, the polar EA (IC 50 = 13.09μg mL -1 for DPPH and 4.80 μg mL -1 for ABTS) and BU (IC 50 = 15.10 μg ml -1 for DPPH and 5.90 μg mL -1 for ABTS) fractions exhibited higher scavenging potential than the non-polar fractions (HE and DC).These results suggested that antioxidant compounds present in crude extract (CE) were concentrated on these two fractions.Moreover, both EA and BU fractions showed IC 50 values similar to the positive controls tested (rutin: 10.74 μg mL -1 and trolox: 2.59 μg mL -1 ).
Regarding the TPC and TF content, as expected, EA (526.80 mg EAG g -1 ; 97.13 mg EQ g -1 ) and BU (426.19 mg EAG g -1 ; 82.07 mg EQ g -1 ) fractions presents the higher levels of phenols and flavonoids than HE and DC fractions.Interestingly, the most polar hydromethanolic fraction (HM) presented lower antioxidant activity, probably due to the grouping of all antioxidant compounds in EA and BU fractions in view of the exhaustive partition process.
In the present study, phenolic compounds (11, 12, 15, 16 and 17) were isolated from the EA fraction of V. nudiflora aerial parts (Figure 1).9][70] Therefore, our results indicated the potential use of polar fractions of V. nudiflora as a natural antioxidant product.

Conclusions
In the present work we investigated the chemical composition, as well as the antioxidant potential and antiproliferative activity of Vernonanthura nudiflora aerial parts.Besides 25 known compounds, some of them identified for the first time in V. nudiflora, one new hirsutinolide derivative was also described.These results contribute to phytochemical knowledge of this species, and suggest that sesquiterpene lactones, especially of hirsutinolide-type, can be considered chemotaxonomic markers of the Vernonanthura genus.The polar fractions (EA and BU) showing higher content of phenolic compounds and flavonoids, presented higher antioxidant activity, with IC 50 values similar to the positive controls.Furthermore, the non-polar fractions (HE and DC) showed the better antiproliferative activity that could be attributed to the presence of hirsutinolide sesquiterpene lactones.The cytostatic effects observed for some isolated sesquiterpene lactones supported this hypothesis.Concluding, these results showed the potential of V. nudiflora aerial parts as a promising source of bioactive molecules for a potential use of as a natural antioxidant, and in the treatment of different types of cancer.

Figure 1 .
Figure 1.Chemical structures of the compounds isolated from V. nudiflora.

Figure 3 .
Figure 3.Chemical structures of the sesquiterpene lactones identified in the dichloromethane fraction by UHPLC-HRMS/MS.

Table 2 .
Data of the compounds identified in the dichloromethane fraction of V. nudiflora by UHPLC-HRMS/MS R : retention time; fragment ions from compounds 18-22 and 26 were not found.