Chemical Constituents from Leaves of Baccharis sphenophylla (Asteraceae) and Their Antioxidant Effects

Baccharis is one of the largest genera of Asteraceae and its species are used in folk medicine for several medicinal purposes due to the presence of bioactive compounds. We investigated the phytochemical composition of polar extracts of B. sphenophylla. Using chromatographic procedures, diterpenoids (ent-kaurenoic acid), flavonoids (hispidulin, eupafolin, isoquercitrin, quercitrin, biorobin, rutin, and vicenin-2), caffeic acid, and chlorogenic acid derivatives (5-O-caffeoylquinic acid and its methyl ester, 3,4-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and 3,5-di-O-caffeoylquinic acid and its methyl ester) were isolated from polar fractions and are described. The extract, polar fractions, and fifteen isolated compounds were evaluated in relation to radical scavenging activity using two assays. Chlorogenic acid derivatives and flavonols exhibited higher antioxidant effects, confirming that B. sphenophylla is an important source of phenolic compounds with antiradical properties.


Introduction
Baccharis L. is one of the largest and most highly diversified genera of Asteraceae found in the New World. The number of species recognized within the genus ranges from 354 to ca. 500 species [1,2]. Approximately 90% of Baccharis species are found in South America, and they are distributed mainly in the warm temperate and tropical regions of Argentina, Brazil, Chile, Colombia, and Mexico. In Brazil, 179 species have been described and, among them, 114 are endemic species [3].
In our continuing efforts to search for bioactive compounds in Baccharis species [24,25], we have focused on B. sphenophylla Dusén ex Malme (Figure 1), a rare and endemic plant found in the highlands of south and southeast Brazil; specifically, in the states of Paraná, São Paulo, and Minas Gerais. Previously, the n-hexane extract of this species was studied, demonstrating a long-chain ester of p-coumaric acid, two sesquiterpenoids, and four diterpenoids with antitrypanosomal effects [26]. Herein, we describe the isolation, structure, In our continuing efforts to search for bioactive compounds in Baccharis species [24,25], we have focused on B. sphenophylla Dusén ex Malme (Figure 1), a rare and endemic plant found in the highlands of south and southeast Brazil; specifically, in the states of Paraná, São Paulo, and Minas Gerais. Previously, the n-hexane extract of this species was studied, demonstrating a long-chain ester of p-coumaric acid, two sesquiterpenoids, and four diterpenoids with antitrypanosomal effects [26]. Herein, we describe the isolation, structure, and antiradical activities of compounds obtained from polar extracts of the leaves of B. sphenophylla.

Structural Elucidation of Compounds
Extracts from leaves of Baccharis sphenophylla (Asteraceae) were obtained with hexane and, subsequently, with ethanol, both until exhaustion. The ethanol extract was partitioned with dichloromethane (DCM), ethyl acetate (EtOAc), and n-butanol (BuOH). The chromatographic fractionation of the DCM fraction allowed the isolation of compounds 1-3 ( Figure 2). Compound 1 exhibited both 1 H and 13 C NMR data compatible with the entkaurenoic acid, a diterpenoid previously isolated from B. sphenophylla [26]. Compounds 2 and 3 showed singlets at δ 6.80 and δ 6.66 in their 1 H NMR spectra, respectively, which were assigned to H-3 from a flavone skeleton. These data were corroborated by the UV spectra, which showed absorption bands at 269 nm and 335 nm and at 269 nm and 345 nm, respectively, both characteristic of flavones [27]. Compound 2 showed two doublets at δ 7.89 (d, J = 8.8 Hz, 2H) and δ 6.92 (d, J = 8.8 Hz, 2H) corresponding to ortho-coupling between H-2′,6′/H-3′,5′ from the 4′-hydroxylated ring B of flavones. On the other hand, compound 3 showed a typical 3',4´-dioxygenated substitution pattern at ring B, exhibiting two doublets at δ 7.40 (d, J = 2.0 Hz, 1H) and δ 6.89 (d, J = 8.0 Hz, 1H) and a doublet of doublets at δ 7.42 (dd, J = 8.0, 2.0 Hz, 1H). Both compounds showed a singlet at δ 6.58, which was assigned to H-8 of the 5,6,7-trioxygenated A-ring, and a methoxyl group was inferred from a singlet at δ 3.75 (s, 3H). Through heteronuclear multiple bond correlations (HMBCs), these groups were bonded at C-6 in both compounds. These data were consistent with the structures of the flavonoids hispidulin (2) and eupafolin (3) (Figure 2), which was subsequently confirmed through a comparison of the spectroscopic data ( 1 H and 13 C NMR) with those reported in the literature [28,29].

Antiradical Properties of the Extract, Fractions, and Compounds
The samples (ethanol extract of Baccharis sphenophylla, three partition fractions, and isolated compounds) were evaluated in relation to their antiradical capacities using DPPH and ABTS assays. Table 1 shows the results obtained for the extract and partition fractions. Although the differences in the values of the antiradical capacities were observed with both methods, the trends for the samples were internally similar (Table 1). Values obtained with the ABTS assay were higher than those obtained with the DPPH assay. Additionally, the antiradical capacity of the ethyl acetate fraction of B. sphenophylla was higher than the ethanol crude extract, as well as the other fractions from this extract.
The isolated compounds were evaluated in both assays, and the results are shown in Table 2. The ent-kaurenoic acid (1) was not able to scavenge the radicals used in either assay; therefore, the IC 50 was not determined (value higher than 200 µmol.L −1 ). The same pattern of response obtained with the mixtures was observed for the isolated compounds; i.e., the values for the antiradical capacity in the ABTS assay were higher than those obtained with the DPPH assay. The ent-kaurenoic acid (1) isolated from the DCM fraction was not able to scavenge the radicals used in either assay; 2 this column describes the source from which the compounds were isolated. DCM, EtOAc, and n-BuOH are, respectively, the dichloromethane, ethyl acetate, and n-butanol fractions. Different letters indicate statistical difference (p < 0.05).

Discussion
This study revealed for the first time the phytochemical composition of the polar extract of Baccharis sphenophylla. Fifteen compounds were isolated and identified and, among them, fourteen are described for the first time for this species. These compounds were diterpenoids, flavonoids, and chlorogenic acid derivatives, which are natural products frequently found in Baccharis species [4,5].
The antiradical capacities of different polar fractions of the plant were evaluated using two assays. The obtained results showed that the antiradical capacities were different in the two assays for the same samples. This fact can be explained by the reactivity of both the generated radicals. The stable free radical DPPH only interacted with more reactive antiradical compounds (constituents with low reactivity found in the mixture are would probably not have been detected by this assay), while the ABTS free radical was able to react with these compounds. Therefore, the differences in the capacity values determined in these assays can be attributed to the presence of low-reactivity antiradical compounds in the samples. A similar pattern of response was observed with the hydroalcoholic crude extracts of Baccharis burchellii and B. crispa analyzed with these assays [35]. The higher antiradical capacity showed by the ethyl acetate fraction can be explained by its chemical composition. This sample was mainly composed of flavonoids and chlorogenic acid derivatives, natural products known for their antiradical activity [13,36].
When analyzing the flavonoids (compounds 2-3 and 11-15), flavonols (11)(12)(13)(14) had a greater ability to scavenge the radicals than the flavones (2-3, 15). The isolated flavones were apigenin (compounds 2 and 15) or luteolin (compound 3) derivatives. The first do not contain the catechol group in their structures, which further decreases their antiradical capacities [37]. Additionally, compounds 2-3 had methoxylated structures, contributing to the decrease in their antiradical capacity. However, the oxygenation of C-3 found in the flavonols contributed to the higher activity observed for these samples. Among the flavonols, those with the catechol group in the B ring (compounds 11-13) had the highest antiradical capacities. For flavonoids, the structural requirements to achieve better radical scavenging activities are: (i) the presence of a catechol group in ring B, which has better electron-donating properties and is a radical target; (ii) a 2,3-double bond conjugated with the 4-oxo group, which is responsible for electron delocalization; and (iii) the presence of a 3-hydroxyl group in the heterocyclic ring, which also increases radical scavenging activity [37]. The flavonoids isolated from the leaves of B. sphenophylla seemed to follow these general trends. Lastly, the different glycosides found in the structures of flavonols (11)(12)(13)(14) had no apparent effects on the antiradical activity.
Chlorogenic acid derivatives are already known for their potent antioxidant capacity because they have a greater capacity to stabilize radicals due to the presence of a catechol group in their structures. Comparing the chlorogenic acid derivatives containing two caffeoyl groups bonded in the quinic acid (compounds 6-9) with those that showed only one caffeoyl group in the structure (compounds 4 and 5), the di-caffeoylquinic acids showed higher antiradical activity. This fact can be justified by the presence of an additional caffeoyl group esterified in the quinic acid ( Table 2). The most active compounds were the 3,4-di-Ocaffeoylquinic acid (6) and 4,5-di-O-caffeoylquinic acid (8), both of them having the caffeoyl group esterified in the C-4 position of the quinic acid. In the literature, the antiradical activity of di-caffeoylquinic acids against DPPH radicals has been analyzed, showing IC 50 values compatible with those reported in this work [30,38]. When comparing the antiradical activity of methyl esters with their respective acids, it was possible to detect an increase in the activity. The higher lipophilicity of methyl esters could have been associated with slightly superior stabilization of both radicals. In summary, among the chlorogenic acids, esterification at the C-4 position seemed more relevant for antiradical activity, additional caffeoyl groups in the structures increased the antiradical activity, and methyl esters derivatives showed a higher capacity for trapping radicals than their respective acids.

General Experimental Procedures
Column chromatography (CC) was performed with a Sephadex LH-20 (GE Healthcare, Chicago, IL, USA) or silica gel 60 (Merck, Darmstadt, Germany). HPLC-grade solvents with the T.J. Baker trademark were used for the HPLC chromatography analyses. Analytical HPLC-DAD-UV analyses were carried out with an Agilent 1260 system (1260 Infinity LC system, Agilent Technologies, La Jolla, CA, USA) equipped with an ultraviolet spectrum scanning detector using an arrangement of photodiodes with a 60 mm flow cell. A Zorbax Eclipse plus a reverse phase C 18 column (4.6 mm × 150 mm, 3.5 µm, Agilent, La Jolla, CA, USA) was used as the stationary phase, and a flow rate of 1.0 mL·min −1 was employed for analysis on an analytical scale with the column temperature set to 45 • C. The injection volume of the sample was 3 µL and the sample was dissolved in methanol at a concentration of 1 mg.mL −1 . For the separation of compounds, an Agilent 1200 semi-preparative chromatography system (1200 LC system, Agilent Technologies, La Jolla, CA, USA) was used with a C 18 Zorbax Eclipse plus an LC-18 column (25 cm × 10 mm, 5 µm, Agilent, La Jolla, CA, USA), a flow rate of 4.176 mL·min −1 for solvents, and a column temperature of 45 • C. The injection volume for the sample was 200 µL and the sample was dissolved in methanol at a concentration of 100 g.L −1 . Both scales (analytical and semi-preparative) were employed as solvents: A-milli-Q water acidified with 0.1% acetic acid (v/v) and B-acetonitrile (ACN).
The nuclear magnetic resonance (NMR) spectra of hydrogen-1 ( 1 H NMR) and carbon-13 ( 13 C NMR) were recorded on a Bruker Avance III 300 Fourier-transform spectrometer (Bruker, Bremen, Germany) equipped with a 5 mm probe and operating at 300.11 MHz for 1 H NMR and 75.5 MHz for 13 C NMR at the Institute of Chemistry of the University of São Paulo. Standard pulse sequences from the Bruker TopspinTM (Bruker, Bremen, Germany) library were used for two-dimensional spectra. Gradient-enhanced sequences were used for the heteronuclear two-dimensional experiments. Chloroform-D, dimethylsulfoxide-d 6
The EtOAc fraction (1.3 g) was subjected to Sephadex LH-20 column chromatography and eluted with methanol to produce six groups (A-F). Groups B (80 mg) and C (59 mg) were subjected to separation using semi-preparative HPLC (same conditions as DCMF; method: 0-3 min: 10 → 20% B; 3-7 min: 25% B) to produce caffeic acid ( The n-butanol fraction (BuF, 1.0 g) was subjected to Sephadex LH-20 column chromatography and eluted with methanol, giving rise to four groups. BuF-1 and BuF-2 showed the presence of compounds 4, 5, and 10, previously isolated and identified in the ethyl acetate fraction. BuF-3 (253.1 mg) yielded vicenin-2 (15, 6,8-di-C-β-glucopyranosylapigenin). The NMR data for the characterization of the isolated compounds are shown in the Supplementary Material.

Antioxidant Assays
Antioxidant assays were performed using a microplate reader, BioTek ® Synergy™ H1 (Agilent Technologies, Santa Clara, CA, USA), with 96-well microplates. Methanol was used as a solvent for the dilutions of the fractions, isolated compounds, Trolox (standard), and negative control. The data analyses were carried out using the software Statistica version 11 (StatSoft, Tulsa, OK, USA).

DPPH Radical Scavenging Assay
The DPPH radical scavenging assay was carried out as described in the literature [35]. Briefly, 3.5 to 3.9 mg of DPPH was dissolved in 50 mL of methanol to prepare the DPPH solution. The exact concentration of the DPPH solution was determined spectrophotometrically with the maximum absorbance at 515 nm (εDPPH = 1.25 × 10 4 L.mol −1 .cm −1 ). The Trolox antiradical solution was prepared with 1.25 mg of the compound dissolved in 2.5 mL of methanol. The prepared solutions were placed in an ultrasonic homogenizer for 5 min to ensure complete solubilization.
Analyses were performed with a microplate reader for absorbance with an optical path of 5 mm and a total volume of 220 µL. Measurements were initiated with the addition of 200 µL DPPH to 20 µL of the sample solution (extract or pure compound with antiradical activity). The kinetics of the reaction was measured from the absorbance of the DPPH solution at 515 nm. All kinetic tests were performed in triplicate with independent measurements, and the results were analyzed and represented as the mean ± standard deviation in the program Origin Pro 8.5 to obtain the kinetic curves.
The variation in absorbance (∆Abs.) between T0 and T50 (AbsTinitial-AbsTfinal) showed a linear correlation with the antiradical concentration. In order to calculate the antiradical activity of the fractions 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, making it possible to obtain the corresponding antiradical capacity as a percentage of Trolox (%Tx) using Equation (1)