Two New Flavonoids from the Leaves of Baccharis oblongifolia (Ruiz and Pav.) Pers. (Asteraceae)

In this work, two new flavonoids, oblongifolioside A (1) and oblongifolioside B (2), along with eight known compounds (3–10), are isolated from the leaves of Baccharis oblongifolia (Asteraceae). The new structures are established through spectroscopic data and the known compounds are identified by comparison with data reported in the literature. The compounds (1–10) are evaluated in relation to their antiradical properties. Compounds 1 and 2 are found to exhibit high antiradical activity compared to their respective non-acylated flavonoids.


Introduction
Baccharis L. (Astereae, Asteraceae) is a large New World genus comprising between 354 and 500 species [1,2]. Approximately 90% of Baccharis species are found in South America and distributed mainly in the warm temperate and tropical regions of Argentina, Brazil, Chile, Colombia, and Mexico. In Brazil, 179 species are found chiefly in the Southern and Southeastern regions. Among them, 115 are endemic species [3] which are restricted to the elevated altitudes of the Atlantic Forest.
In our continuing efforts in the search for new bioactive compounds from Brazilian Asteraceae species, this paper describes two new acylated flavonoids, oblongifolioside A (1) and oblongifolioside B (2), and eight known compounds (3-10) from B. oblongifolia leaves, as shown in Figure 1. The new structures were elucidated by spectroscopic data and the known ones were identified by comparison with literature data. Herein, we report the isolation, structure elucidation, and antiradical activities of these compounds.

Structural Elucidation
Oblongifolioside A (1) was isolated as a yellow powder. The molecular formula of 1 was assigned C36H36O19 based on its HRESIMS at m/z 771.1772 [M − H] − , indicating 19 degrees of unsaturation. The UV spectrum exhibited absorption maxima at 254 and 345 nm, with a shoulder at
The 13 C NMR spectrum displayed thirty-six carbon signals, including a carbonyl, a carboxyl, twelve quaternary carbons, ten unsaturated methines, ten oxymethines, one oxygenated methylene, and one methyl group. Through HMBC correlations, it was possible to establish the connectivity among groups in the structure. The signals at δ 5.48 and δ 5.04 attributed to H-1 and H-2 from the glycosyl moiety correlated with the carbons at δ 133.3 and δ 167.2, respectively, which were attributed to C-3 from quercetin and to C-9 from a caffeoyl group. These correlations confirmed the glucose at C-3 of quercetin and the caffeoyl group esterified on hydroxyl at C-2 of glucose. Other key HMBC correlations are shown in Figure 2. With the aid of HSQC and HMBC experiments, all 1 H and 13 C NMR signals of 1 were assigned as shown in Table   suggested the presence of a substituent group at C-2′' from the glycosyl moiety (Figures S1 and S2). The 13 C NMR spectrum displayed thirty-six carbon signals, including a carbonyl, a carboxyl, twelve quaternary carbons, ten unsaturated methines, ten oxymethines, one oxygenated methylene, and one methyl group. Through HMBC correlations, it was possible to establish the connectivity among groups in the structure. The signals at δ 5.48 and δ 5.04 attributed to H-1′' and H-2′' from the glycosyl moiety correlated with the carbons at δ 133.3 and δ 167.2, respectively, which were attributed to C-3 from quercetin and to C-9′''' from a caffeoyl group. These correlations confirmed the glucose at C-3 of quercetin and the caffeoyl group esterified on hydroxyl at C-2′' of glucose. Other key HMBC correlations are shown in Figure 2. With the aid of HSQC and HMBC experiments, all 1 H and 13 C NMR signals of 1 were assigned as shown in Table 13 C and HMBC NMR data of 1 and 2 (δ in ppm, J in Hz, CD3OD).   13 C and HMBC NMR data of 1 and 2 (δ in ppm, J in Hz, CD 3 OD).  The 13 C-NMR spectrum displayed thirty-four carbon signals, including a carbonyl, a carboxyl, twelve quaternary carbons, eight unsaturated methines, ten oxymethines, one oxygenated methylene, and one methyl group. Through HMBC correlations, it was possible to establish the connectivity among groups in the structure. The signals at δ 5.55 and δ 5.01 attributed to H-1 and H-2 of the glycosyl moiety correlated with the carbons at δ 133.3 and δ 167.1, respectively, which were attributed to C-3 from kaempferol and to C-9 from a caffeoyl group, confirming the bond of glucose at C-3 of kaempferol and the caffeoyl group esterified on hydroxyl at C-2 of glucose. Other key HMBC correlations are shown in Figure 2 and other signals of compound 2 were assigned and are shown in Table 1.

General Experimental Procedures
Column chromatography (CC) was performed on a Sephadex LH-20 (GE Healthcare). HPLC grade solvents of the trademark T.J. Baker were used for the HPLC chromatography analyses. Analytical HPLC analyses were carried out on an Agilent 1260 system (G1311 pump 110 and G1315D photodiode array detector; Palo Alto, US) with a 60 mm flow cell. Zorbax Eclipse plus reverse phase C18 (4.6 mm × 150 mm, 3.5 µm, Agilent) was used as the stationary phase and a flow rate of 1.0 mL·min −1 was employed for analyses on the analytical scale. For separation of compounds the Agilent 1200 semi-preparative chromatograph system (Palo Alto, US) was used with a C18 Zorbax eclipse plus LC-18 column (25 cm × 10 mm) with 5 µm diameter particles and a flow rate of 4.176 mL·min −1 of solvent A: milli-Q water acidified with 0.1% acetic acid (v/v) and solvent B: acetonitrile (ACN). The column temperature was 45 • C, the injection volume of the sample was 200 µL and the sample was dissolved in methanol at a concentration of 100 g·L −1 . NMR spectra of hydrogen-1 ( 1 H-NMR) and carbon-13 ( 13 C-NMR) were recorded on a Bruker AIII 500 MHz spectrometer (MA, US) operating at 500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR at the Institute of Chemistry of the University of São Paulo. The spectra were obtained in deuterated methanol from Sigma-Aldrich as a solvent. NMR data were processed using MestreNova 9.0 software. Optical rotations were measured on a Perkin Elmer 243B polarimeter. Mass spectra were recorded on an Amazon ETD Bruker Daltonics (MA, US) with capillary 4500V and nebulizer at 27 psi in a negative mode.

Antiradical Assay
Evaluation of antiradical activity was performed according to a protocol published in the literature [32]. Briefly, the DPPH solution was prepared from 3.5 mg to 3.9 mg of DPPH in 50 mL of methanol. The exact concentration of the DPPH solution was determined spectrophotometrically by the maximum absorbance at 515 nm (ε DPPH = 1.25 × 10 4 L·mol −1 ·cm −1 ). The Trolox antiradical solution was prepared with 1.25 mg in 2.5 mL methanol. The solutions prepared remained for 5 min in a sonicator for complete solubilization.
Analyses were performed on a microplate reader for absorbance with an optical path of 5 mm with a total volume of 220 µL. Measurements were initiated by the addition of 200 µL DPPH in 20 µL of the sample solution (pure compound). The kinetics of the reactions were observed by absorbance of the DPPH solution at 515 nm. All kinetic tests were performed in triplicate in independent measurements and the results treated and represented with mean ± standard deviation in the program Origin Pro 8.5 to obtain the kinetic curves.
The variation of absorbance (∆Abs.) between T0 and T50 (AbsTinitial-AbsTfinal) shows a linear correlation with the antiradical concentration. In order to calculate the antiradical activity of the phases and pure compounds, the angular coefficients (α) of the antiradical (A) and Trolox (T) standard deviations were used as a function of the absorbance variation in order to obtain the corresponding antiradical capacity as a percentage of Trolox (%Tx), according to Equation (1) [32]: %Tx = (αA/αT) × 100 (1) In addition to correlating the concentration of the antiradical compound with the variation of the absorbance, the correlation of this concentration was also made directly with the concentration of the DPPH radical consumed. From this correlation it was possible to directly obtain the number of DPPH radicals sequestered per antiradical molecule.
The percentage of antiradical activity was calculated from Equation (2)